US20160298096A1 - Crispr-cas system materials and methods - Google Patents

Crispr-cas system materials and methods Download PDF

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US20160298096A1
US20160298096A1 US15/037,371 US201415037371A US2016298096A1 US 20160298096 A1 US20160298096 A1 US 20160298096A1 US 201415037371 A US201415037371 A US 201415037371A US 2016298096 A1 US2016298096 A1 US 2016298096A1
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dna
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Emmanuelle Charpentier
Krzysztof Chylinski
Ines Fontara
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CRISPR Therapeutics AG
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Definitions

  • the invention relates to type II CRISPR-Cas systems of Cas9 enzymes, guide RNAs and associated specific PAMs.
  • CRISPR-Cas Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated proteins
  • trans-activating CRISPR RNA (tracrRNA) (15,16) binds to the invariable repeats of precursor CRISPR RNA (pre-crRNA) forming a dual-RNA (14-17) that is essential for both RNA co-maturation by RNase III in the presence of Cas9 (15-17), and invading DNA cleavage by Cas9 (14,15,17-19).
  • Cas9 guided by the duplex formed between mature activating tracrRNA and targeting crRNA introduces site-specific double-stranded DNA (dsDNA) breaks in the invading cognate DNA (14,17-19).
  • Cas9 is a multi-domain enzyme (14,20,21) that uses an HNH nuclease domain to cleave the target strand (defined as complementary to the spacer sequence of crRNA) and a RuvC-like domain to cleave the non-target strand (14,22,23), enabling the conversion of the dsDNA cleaving Cas9 into a nickase by selective motif inactivation (2,8,14,24,25).
  • DNA cleavage specificity is determined by two parameters: the variable, spacer-derived sequence of crRNA targeting the protospacer sequence (a protospacer is defined as the sequence on the DNA target that is complementary to the spacer of crRNA) and a short sequence, the Protospacer Adjacent Motif (PAM), located immediately downstream of the protospacer on the non-target DNA strand (14,18,23,26-28).
  • a protospacer is defined as the sequence on the DNA target that is complementary to the spacer of crRNA
  • PAM Protospacer Adjacent Motif
  • RNA-guided Cas9 can be employed as an efficient genome editing tool in human cells (1,2,8,11), mice (9,10), zebrafish (6), drosophila (5), worms (4), plants (12,13), yeast (3) and bacteria (7).
  • the system is versatile, enabling multiplex genome engineering by programming Cas9 to edit several sites in a genome simultaneously by simply using multiple guide RNAs (2,7,8,10).
  • the easy conversion of Cas9 into a nickase was shown to facilitate homology-directed repair in mammalian genomes with reduced mutagenic activity (2,8,24,25).
  • the DNA-binding activity of a Cas9 catalytic inactive mutant has been exploited to engineer RNA-programmable transcriptional silencing and activating devices (29,30).
  • RNA-guided Cas9 from S. pyogenes, Streptococcus thermophilus, Neisseria meningitidis and Treponema denticola have been described as tools for genome manipulation (1-13,24,25,31-34 and Esvelt et al. PMID: 24076762).
  • the present invention expands the RNA-programmable Cas9 toolbox to additional orthologous systems.
  • the diversity and interchangeability of dual-RNA:Cas9 in eight representatives of phylogenetically defined type II CRISPR-Cas groups was examined herein.
  • the results of this work not only introduce a wider range of Cas9 enzymes, guide RNA structures and associated specific PAMs but also enlighten the evolutionary aspects of type II CRISPR-Cas systems, including coevolution and horizontal transfer of the system components.
  • the present disclosure provides guide RNAs, both single-molecule and double-molecule guide RNAs, as well as methods for manipulating DNA in a cell using the guide RNAs and/or DNAs (including vectors) encoding the guide RNAs.
  • Complexes comprising the guide RNAs and Cas9 endonucleases are also provided.
  • the single-molecule guide RNAs comprise a DNA-targeting segment and a protein-binding segment, wherein the protein-binding segment comprises a tracrRNA set out in Supplementary Table S5 or wherein the protein-binding segment comprises a tracrRNA at least 80% identical over at least 20 nucleotides to a tracrRNA set out in Supplementary Table S5.
  • the protein-binding segment comprises a CRISPR repeat set out in Supplementary Table S5 that is the CRISPR repeat cognate to the tracrRNA of the protein-binding segment.
  • the DNA-targeting segment comprises RNA complementary to a protospacer-like sequence in a target DNA 5′ to a PAM sequence.
  • the tracrRNA and CRISPR repeat are respectively the C. jejuni tracrRNA and its cognate CRISPR repeat set out in Supplementary Table S5 and the PAM sequence is NNNNACA. In some embodiments, the tracrRNA and CRISPR repeat are respectively at least 80% identical to the C. jejuni tracrRNA and its cognate CRISPR repeat set out in Supplementary Table S5 and the PAM sequence is NNNNACA.
  • the single-molecule guide RNA comprises a sequence that hybridizes to a protospacer-like sequence set out in one of SEQ ID NOs: 801-2701.
  • the disclosure provides a DNA encoding a single-molecule guide RNA of the invention.
  • the disclosure provides a vector comprising a DNA encoding a single-molecule guide RNA of the invention.
  • the disclosure provides a cell comprising a DNA encoding a single-molecule guide RNA of the invention.
  • the disclosure provides a double-molecule guide RNA comprising: a targeter-RNA and an activator-RNA complementary thereto, wherein the activator-RNA comprises a tracrRNA set out in Supplementary Table S5 or wherein the activator-RNA comprises a tracrRNA at least 80% identical over at least 20 nucleotides to a tracrRNA set out in Supplementary Table S5.
  • the double-molecule guide RNA comprises a modified backbone, a non-natural internucleoside linkage, a nucleic acid mimetic, a modified sugar moiety, a base modification, a modification or sequence that provides for modified or regulated stability, a modification or sequence that provides for subcellular tracking, a modification or sequence that provides for tracking, or a modification or sequence that provides for a binding site for a protein or protein complex.
  • the targeter-RNA comprises a CRISPR repeat set out in Supplementary Table S5.
  • the targeter-RNA comprises a CRISPR repeat set out in Supplementary Table S5 that is the cognate CRISPR repeat of the tracrRNA of the activator-RNA.
  • the targeter-RNA further comprises RNA complementary to a protospacer-like sequence in a target DNA 5′ to a PAM sequence.
  • the tracrRNA and CRISPR repeat are respectively the C. jejuni tracrRNA and its cognate CRISPR repeat set out in Supplementary Table S5 and the PAM sequence is NNNNACA.
  • the tracrRNA and CRISPR repeat are at least 80% identical to respectively the C. jejuni tracrRNA and its cognate CRISPR repeat set out in Supplementary Table S5 and the PAM sequence is NNNNACA.
  • the double-molecule guide RNA comprises a sequence that hybridizes to a protospacer-like sequence set out in one of SEQ ID NOs: 801-2701.
  • the disclosure provides a DNA encoding a double-molecule guide RNA of the invention.
  • the disclosure provides a vector comprising a DNA encoding a double-molecule guide RNA of the invention.
  • the disclosure provides a cell comprising a DNA encoding a double-molecule guide RNA of the invention.
  • the disclosure provides methods for manipulating DNA in a cell, comprising contacting the DNA with a Cas9 ortholog-guideRNA complex, wherein the complex comprises: (a) a C. jejuni Cas9 endonuclease heterologous to the cell or an endonuclease with an activity portion at least 90% identical to the activity portion of the C. jejuni Cas9 endonuclease, and a guide RNA targeting the complex to a protospacer-like sequence in the DNA 5′ to the PAM sequence NNNNACA; (b) a P. multocida Cas9 endonuclease heterologous to the cell or an endonuclease with an activity portion at least 90% identical to the activity portion of the P.
  • the guide is a single-molecule guide RNA. In some embodiments, the guide RNA is a double-molecule guide RNA.
  • the protospacer-like sequence targeted is in a CCR5, CXCR4, KRT5, KRT14, PLEC or COL7A1 gene.
  • the protospacer-like sequence is in a chronic granulomatous disease (CGD)-related gene CYBA, CYBB, NCF1, NCF2 or NCF4.
  • CCD chronic granulomatous disease
  • the protospacer-like sequence targeted is in a gene encoding B-cell lymphoma/leukemia IIA (BCL11A) protein, an erythroid enhancer of BCL11A or a BCL11A binding site.
  • the protospacer-like sequence targeted is up to 1000 nucleotides upstream of the above mentioned genes.
  • the guide RNA comprises a sequence complementary to a protospacer-like sequence set out in one of SEQ ID NOs: 801-2701.
  • the disclosure provides a recombinant vector encoding: (a) a guide RNA, wherein the guide RNA comprises a DNA-targeting segment complementary to a protospacer-like sequence in the DNA 5′ to the PAM sequence NNNNACA; and (b) a C. jejuni Cas9 endonuclease (for example, set out in SEQ ID NO: 50) or an endonuclease with an activity portion at least 90% identical to the activity portion of the C. jejuni Cas9 endonuclease.
  • the DNA-targeting segment complementary to the protospacer-like sequence is RNA complementary to the target sequences set out in one of SEQ ID NOs: 801-973, 1079-1222, 1313-1348, 1372-1415, 1444-1900, 2163-2482 or 2667-2686.
  • Methods of using the vectors to manipulate DNA in a cell are also provided.
  • the disclosure provides a recombinant vector encoding: (a) a guide RNA, wherein the guide RNA comprises a DNA-targeting segment complementary to a protospacer-like sequence in the DNA 5′ to the PAM sequence GNNNCNNA or NNNNC; and (b) a P. multocida Cas9 endonuclease (for example, set out in SEQ ID NO: 1) or an endonuclease with an activity portion at least 90% identical to the activity portion of the P. multocida Cas9 endonuclease.
  • a guide RNA comprises a DNA-targeting segment complementary to a protospacer-like sequence in the DNA 5′ to the PAM sequence GNNNCNNA or NNNNC
  • a P. multocida Cas9 endonuclease for example, set out in SEQ ID NO: 1
  • the DNA-targeting segment complementary to the protospacer-like sequence is RNA complementary to the target sequences set out in one of SEQ ID NOs:974-1078, 1223-1312, 1349-1371, 1416-1443, 1901-2162, 2483-2666 or 2687-2701. Methods of using the vectors to manipulate DNA in a cell are also provided.
  • the disclosure provides a recombinant vector encoding: (a) a guide RNA, wherein the guide RNA comprises a DNA-targeting segment complementary to a protospacer-like sequence in the DNA 5′ to the PAM sequence NG; and (b) a F. novicida Cas9 endonuclease (fore example, set out in SEQ ID NO: 43) or an endonuclease with an activity portion at least 90% identical to the activity portion of the F. novicida Cas9 endonuclease.
  • Methods of using the vectors to manipulate DNA in a cell are also provided.
  • the disclosure provides a recombinant vector encoding: (a) a guide RNA, wherein the guide RNA comprises a DNA-targeting segment complementary to a protospacer-like sequence in the DNA 5′ to the PAM sequence NNAAAAW; and (b) a S. thermophilus ** Cas9 endonuclease or an endonuclease with an activity portion at least 90% identical to the activity portion of the S. thermophilus ** Cas9 endonuclease.
  • Methods of using the vectors to manipulate DNA in a cell are also provided.
  • the disclosure provides a recombinant vector encoding: (a) a guide RNA, wherein the guide RNA comprises a DNA-targeting segment complementary to a protospacer-like sequence in the DNA 5′ to the PAM sequence NGG; and (b) a L. innocua Cas9 endonuclease (for example, set out in SEQ ID NO: 3) or an endonuclease with an activity portion at least 90% identical to the activity portion of the L. innocua Cas9 endonuclease.
  • Methods of using the vectors to manipulate DNA in a cell are also provided.
  • the disclosure provides a recombinant vector encoding: (a) a guide RNA, wherein the guide RNA comprises a DNA-targeting segment complementary to a protospacer-like sequence in the DNA 5′ to the PAM sequence NGG; and (b) a S. dysgalactiae Cas9 endonuclease (for example, set out in SEQ ID NO: 105) or an endonuclease with an activity portion at least 90% identical to the activity portion of the S. dysgalactiae Cas9 endonuclease.
  • the guide RNA comprises a sequence complementary to a protospacer-like sequence set out in one of SEQ ID NOs: 801-2701.
  • the disclosure provides a method comprising (a) identifying at least 7-20 bases of mammalian genomic DNA adjacent to any of the preceding protospacer-like sequences, and (b) manipulating the mammalian genomic DNA sequence by contacting a mammalian cell with, or administering to a mammal, (i) a DNA-targeting segment complementary to the DNA sequence identified in step (a) and (ii) a protein-binding segment, or nucleic acid(s) encoding (i) and (ii), and (iii) a cas9 endonuclease or a nucleic acid encoding said cas9 endonuclease; and (c) detecting cleavage of the mammalian genomic DNA.
  • the disclosure provides a modified Cas9 endonuclease, modified from any of the Cas9 orthologs disclosed herein, comprising one or more mutations corresponding to S. pyogenes Cas9 mutation E762A, HH983AA or D986A.
  • the modified Cas 9 endonuclease further comprises one or more mutations corresponding to S. pyogenes Cas9 mutation D10A, H840A, G12A, G17A, N854A, N863A, N982A or A984A.
  • the disclosure provides a method for manipulating DNA in a cell, comprising contacting the DNA with a Cas9 ortholog-guide RNA complex, wherein the complex comprises: (a) a Cas9 endonuclease heterologous to the cell and (b) a cognate guide RNA of the Cas9 endonuclease comprising a tracrRNA set out in Supplementary Table S5 or a guide RNA comprising a tracrRNA at least 80% identical to a cognate tracrRNA set out in Supplementary Table S5 over at least 20 nucleotides.
  • the guide is a single-molecule guide RNA.
  • the guide RNA is a double-molecule guide RNA.
  • the guide RNA comprises a sequence complementary to a protospacer-like sequence set out in one of SEQ ID NOs: 801-2701. Complexes used in the methods are also provided.
  • the disclosure provides a method for manipulating DNA in a cell, comprising contacting the DNA with a Cas9 ortholog-guide RNA complex, wherein the complex comprises: (a) a cognate guide RNA for a first Cas9 endonuclease from a cluster in Supplementary Table S2 and (b) a second Cas9 endonuclease from the same cluster that is exchangeable with preserved high cleavage efficiency with the first endonuclease and shares at least 80% identity with the first endonuclease over 80% of their length.
  • the guide is a single-molecule guide RNA.
  • the guide RNA is a double-molecule guide RNA.
  • the first Cas9 endonuclease is from S. pyogenes and the second Cas9 endonuclease is from S. mutans .
  • the first Cas9 endonuclease is from S. thermophilus * and the second Cas9 endonuclease is from S. mutans .
  • the first Cas9 endonuclease is from N. meningitidis and the second Cas9 endonuclease is from P. multocida .
  • Complexes used in the methods are also provided.
  • the disclosure provides a method for manipulating DNA in a cell, comprising contacting the DNA with a Cas9 ortholog-guide RNA complex, wherein the complex comprises: (a) a cognate guide RNA of a first Cas9 endonuclease from a cluster in Supplementary Table S6 and (b) an Cas9 endonuclease from the same cluster in Supplementary Table S6 that is exchangeable with the same or lowered cleavage efficiency with the first endonuclease and shares at least 50% amino acid sequence identity with the first endonuclease over 70% of their length.
  • the guide is a single-molecule guide RNA.
  • the guide RNA is a double-molecule guide RNA.
  • the first Cas9 endonuclease is from C. Jejuni and the second Cas9 endonuclease is from P. multocida .
  • the first Cas9 endonuclease is from N. meningitidis and the second Cas9 endonuclease is from P. multocida .
  • Complexes used in the methods are also provided.
  • the disclosure provides a method for manipulating DNA in a cell, comprising contacting the DNA with two or more Cas9-guide RNA complexes, wherein each Cas9-guideRNA complex comprises: (a) a Cas9 endonuclease from a different cluster in Supplementary Table S6 exhibiting less than 50% amino acid sequence identity with the other endonucleases of the method over 70% of their length, and (b) a guide RNA specifically complexed with each Cas9 endonuclease.
  • the guide is a single-molecule guide RNA.
  • the guide RNA is a double-molecule guide RNA.
  • the Cas9 endonucleases are from F.
  • the Cas9 endonucleases are from N. meningitidis and S. mutans .
  • the Cas9 endonucleases are the S. thermophilus * Cas9 and the S. thermophilus ** Cas9. Complexes used in the methods are also provided.
  • the DNA targeted in the cell is a CCR5, CXCR4, KRT5, KRT14, PLEC or COL7A1 gene.
  • the DNA targeted in the cell is a chronic granulomatous disease (CGD)-related gene CYBA, CYBB, NCF1, NCF2 or NCF4.
  • CCD chronic granulomatous disease
  • the protospacer-like sequence targeted is in a gene encoding B-cell lymphoma/leukemia IIA (BCL11A) protein, an erythroid enhancer of BCL11A or a BCL11A binding site.
  • the protospacer-like sequence targeted is up to 1000 nucleotides upstream of the above mentioned genes.
  • the guide RNA comprises a sequence complementary to a protospacer-like sequence set out in one of SEQ ID NOs: 801-2701.
  • FIG. 1 Phylogeny of representative Cas9 orthologs and schematic representation of selected bacterial type II CRISPR-Cas systems.
  • A Phylogenetic tree of Cas9 reconstructed from selected, informative positions of representative Cas9 orthologs multiple sequence alignment is shown (see Supplementary FIG. S2 and Supplementary Table S2).
  • the Cas9 orthologs of the subtypes classified as II-A, II-B and II-C are highlighted with shaded boxes.
  • the colored branches group distinct proteins of closely related loci with similar locus architecture (15). Each protein is represented by the GenInfo (GI) identifier followed by the bacterial strain name. The bootstrap values are given for each node (see Materials and Methods).
  • GenInfo GI
  • Type II CRISPR-Cas loci can differ in the cas gene composition, mostly with cas9, cas1 and cas2 being the minimal set of genes (type II-C, blue), sometimes accompanied with a fourth gene csn2a/b (type II-A, yellow and orange) or cas4 (type II-B, green).
  • the CRISPR array can be transcribed in the same (type II-A, yellow and orange) or in the opposite (types II-B and C, blue and green) direction of the cas operon.
  • tracrRNA and the direction of its transcription differ within the groups (compare type II-A of S. thermophilus ** with type II-A from the other species indicated here (yellow) and compare type II-C of C. jejuni with type II-C of N. meningitidis and P. multocida (blue)).
  • FIG. 2 RNase III is a general executioner of tracrRNA:pre-crRNA processing in type II CRISPR-Cas.
  • RNA sizes in nt and schematic representations of tracrRNA (red-black) and crRNA (green-black) are indicated on the right (16). The vertical black arrows indicate the processing sites.
  • tracrRNA-171 nt and tracrRNA-89 nt forms correspond to primary tracrRNA transcripts.
  • the presence of tracrRNA-75 nt and crRNA 39-42 nt forms indicates tracrRNA and pre-crRNA co-processing.
  • S. pyogenes tracrRNA and pre-crRNA are co-processed by all analyzed RNase III orthologs. The truncated version and catalytic inactive mutant of S. pyogenes RNase III are both deficient in tracrRNA:pre-crRNA processing.
  • FIG. 3 conserved motifs of Cas9 are required for DNA interference but not for dual-RNA processing by RNase III.
  • A Schematic representation of S. pyogenes Cas9. The conserved HNH and splitted RuvC motifs and analyzed amino acids are indicated.
  • B Northern blot analysis of total RNA from S. pyogenes WT, ⁇ cas9 and ⁇ cas9 complemented with pEC342 or pEC342 containing cas9 WT or mutant genes, probed for tracrRNA and crRNA repeat.
  • C In vivo protospacer targeting. Transformation assays of S. pyogenes WT and ⁇ cas9 with pEC85 (vector), pEC85 ⁇ cas9 (cas9), pEC85 ⁇ speM (speM), and pEC85 ⁇ tracrRNA-171 nt plasmids containing speM and cas9 mutants.
  • the CFUs colony forming units per ⁇ g of plasmid DNA were determined in at least three independent experiments.
  • FIG. 4 Cas9 from closely related CRISPR-Cas systems can substitute the role of S. pyogenes Cas9 in RNA processing by RNase III.
  • A Schematic representation of Cas9 from selected bacterial species. The protein sizes and distances between conserved motifs (RuvC and HNH) are drawn in scale. See Supplementary FIG. S1 .
  • B Northern blot analysis of total RNA extracted from S. pyogenes WT, ⁇ cas9 and ⁇ cas9 complemented with pEC342 (backbone vector containing tracrRNA-171 nt and the cas operon promoter from S.
  • pyogenes or pEC342-based plasmids containing cas9 orthologous genes, probed for tracrRNA and crRNA repeat. Mature forms of S. pyogenes tracrRNA and pre-crRNA are observed only in the presence of S. pyogenes Cas9 WT or closely related Cas9 orthologs from S. mutans and S. thermophilus*.
  • FIG. 5 Cas9 orthologs cleave DNA in the presence of their cognate dual-RNA and specific PAM in vitro.
  • A Logo plot of protospacer adjacent sequences derived from BLAST analysis of spacer sequences for selected bacterial species. The logo plot gives graphical representation of most abundant nucleotides downstream of the protospacer sequence. The numbers in brackets correspond to the number of analyzed protospacers.
  • B DNA substrates designed for specific PAM verification. Based on the logo plot for each species, plasmid DNA substrates were designed to contain the speM protospacer and the indicated sequence downstream, either comprising (PAM+) or not (PAM ⁇ ) the proposed PAM.
  • Each Cas9 ortholog in complex with its cognate dual-RNA cleaves plasmids containing the corresponding species-specific PAM (PAM+). No cleavage is observed with plasmids that did not contain the specific PAM (PAM ⁇ ).
  • PAM+ species-specific PAM
  • PAM ⁇ species-specific PAM
  • li linear cleavage product
  • sc supercoiled plasmid DNA.
  • FIG. 6 Cas9 and dual-RNA co-evolved.
  • A In vitro plasmid cleavage assays using S. pyogenes Cas9 in complex with orthologous dual-RNA (upper panel) and orthologous Cas9 enzymes in complex with S. pyogenes dual-RNA (lower panel). Plasmid DNA containing protospacer speM and S. pyogenes PAM (NGG) was incubated with different dual-RNAs in complex with S. pyogenes Cas9. tracrRNA and crRNA-repeat sequences of the dual-RNAs are from the indicated bacterial species, with crRNA spacer targeting speM.
  • plasmid DNA containing speM protospacer and the specific PAM was incubated with Cas9 orthologs in complex with S. pyogenes dual-RNA.
  • S. pyogenes Cas9 can cleave plasmid DNA only in the presence of dual-RNA from S. pyogenes, S. mutans and S. thermophilus * (yellow).
  • Dual-RNA from S. pyogenes can mediate DNA cleavage only with Cas9 from S. pyogenes, S. mutans and S. thermophilus * (yellow).
  • li linear cleavage product
  • sc supercoiled plasmid DNA.
  • FIG. 5 Summary of Cas9 and dual-RNA orthologs exchangeability. Specific PAM sequences were used according to FIG. 5 .
  • the color code reflects the type II CRISPR-Cas subgroups ( FIG. 1 ). +++: 100-75% cleavage activity; ++: 75-50% cleavage activity; +: 50-25% cleavage activity; -: 25-0% cleavage activity observed under the conditions tested.
  • Cas9 and dual-RNA duplexes from the same type II group can be interchanged and still mediate plasmid cleavage providing that the PAM sequence is specific for Cas9. See also Supplementary FIG. S10 .
  • FIG. S1 Biochemical characteristics and SDS-PAGE analysis of Cas9 proteins purified in this study.
  • A Overview of characteristics of Cas9 orthologous proteins allote that the biochemical characteristics of S. pyogenes Cas9 WT and mutants are identical; b GenInfo (GI) Identifier; c ⁇ , Extinction coefficient.
  • B SDS PAGE analysis of purified mutants of Cas9 from S. pyogenes .
  • C SDS PAGE analysis of purified Cas9 orthologs.
  • M PageRulerTM Unstained Protein Ladder (Thermo Scientific).
  • FIG. S2 Multiple sequence alignment of representative Cas9 sequences (see Supplementary Table S2 and Material and Methods).
  • the rows described as Jnet with following GI identifier of a selected Cas9 sequence provide the predicted secondary structure of Cas9 within the corresponding subgroups (sequences indicated below each Jnet). conserveed motifs are marked below the alignment and the mutated amino acid residues are highlighted. Asterisks indicate informative positions chosen for the Cas9 tree reconstruction.
  • FIG. S3 Multiple sequence alignment of representative Cas1 sequences (see Supplementary Table S2 and Materials and Methods). Informative positions chosen for the Cas1 tree reconstruction are marked with asterisks at the bottom of the alignment.
  • FIG. S4 Phylogenetic analysis of representative Cas9 and Cas1 sequences. Phylogenetic trees of Cas1 (left) and Cas9 (right) reconstructed from selected, informative positions of Cas1 and Cas9 multiple sequence alignments are shown (see FIG. 1 and Supplementary FIG. S2 and S 3 ). The Cas1 tree is rooted to the outgroup of selected Cas1 orthologs of type I CRISPR-Cas systems. The Cas1 and Cas9 orthologs of the types classified as II-A, II-B and II-C are highlighted with shaded boxes. The same branch colors were used for each bacterial strain on both trees. Each protein is represented by the GenInfo (GI) identifier followed by the bacterial strain name.
  • GenInfo GI
  • the bootstrap values are given for each node (see Materials and Methods).
  • the scale bars for the branch length are given as the estimated number of amino acid substitution per site. Note the similarity of the trees topology and monophyletic clusters of subtypes II-A and II-B on both trees supported by high bootstrap values.
  • RNase III is a general executioner of tracrRNA:pre-crRNA processing in type II CRISPR-Cas.
  • the dashed-line boxes represented below the Northern blots in (B) show the area of the blots with enhanced exposure. All RNAse III orthologs can co-process S. pyogenes tracrRNA and pre-crRNA. No mature forms of tracrRNA and crRNAs could be observed in ⁇ rnc complemented with the truncated version or catalytically inactive (dead) mutant of RNase IIII.
  • FIG. S6 Multiple sequence alignment of bacterial endoribonucleases III used in the study. Domains indicated below the alignment are according to the domains identified in RNase III from E. coli (58, 59). The conserved catalytic aspartate residue mutated in the catalytically inactive “mc dead” mutant and the last amino acid of the truncated mc mutant are indicated above the alignment with an asterisk and an arrow, respectively.
  • FIG. S7 conserved catalytic amino acid residues of Cas9 are not involved in dual-RNA processing by RNase III.
  • tracrRNA:crRNA co-processing is observed in all strains encoding Cas9 point mutants. Note that in a previous study, we observed low abundance of tracrRNA in the cas9 deletion mutant (16). For this reason, plasmids used in cas9 complementation studies were designed to encode tracrRNA in addition to cas9.
  • FIG. S8 Cas9 and tracrRNA:crRNA co-evolved.
  • A tracrRNA or (B) crRNA repeat probe
  • CRISPR3 CRISPR3
  • FIG. S9 Cas9 orthologs cleave plasmid DNA in the presence of their cognate dual-RNA and specific PAM. Agarose gel electrophoresis analysis of dual-RNA:Cas9 titration (0-100 nM dual-RNA-Cas9 complex) on plasmid DNA (5 nM) containing speM protospacer and adjacent WT PAM (PAM+), imperfect PAM (PAM ⁇ ) or no PAM (PAM ⁇ ).
  • PAM speM protospacer and adjacent WT PAM
  • PAM ⁇ imperfect PAM
  • PAM ⁇ no PAM
  • S. pyogenes S. mutans
  • S. thermophilus* S. thermophilus **
  • N. meningitidis the PAM sequence has already been published (27,28,53,54).
  • PAMs were predicted based on the downstream sequence of protospacer identified in the investigated or related strains (see Supplementary Table S2 and Materials and Methods).
  • the 10 bp sequence located directly downstream of the crRNA-targeted speM protospacer is shown.
  • the nucleotide(s) predicted to belong to the PAM sequence are shaded in grey.
  • li linear cleavage product
  • sc supercoiled plasmid DNA
  • M 1 kb DNA ladder.
  • FIG. S10 Summary of in vitro plasmid cleavage assays of Cas9 orthologs in combination with dual-RNAs. Agarose gel electrophoresis of cleavage assays.
  • A S. mutans Cas9 (50 nM)
  • B S. thermophilus * Cas9 (25 nM)
  • C S. thermophilus ** Cas9 (100 nM)
  • D C. jejuni Cas9 (100 nM)
  • E N. meningitidis Cas9 (100 nM)
  • F P. multocida Cas9 (25 nM)
  • G F.
  • novicida Cas9 100 nM in complex with equimolar concentrations of each of the dual-RNA orthologs were incubated with plasmid DNA (5 nM) containing speM protospacer sequence and the PAM sequence specific to the Cas9 ortholog analyzed.
  • plasmid DNA 5 nM
  • speM protospacer sequence the PAM sequence specific to the Cas9 ortholog analyzed.
  • li linear cleavage product
  • sc supercoiled plasmid DNA
  • M 1 kb DNA ladder.
  • FIG. S11 Cas9 tree topology suggests both horizontal and vertical transfer of type II CRISPR-Cas systems. See FIG. 1 , Supplementary FIG. S4 and Supplementary Table S4. The codes for taxonomy (phyla in color) and habitat (symbols) of the bacterial strains harbouring representative Cas9 orthologs are indicated (right panel).
  • Clusters 2, 4 and 5 group closely related bacteria isolated from diverse habitats indicating vertical transfer of the systems.
  • tracrRNA:crRNA repeat duplexes form similar secondary structures in loci with closely related Cas9 orthologs. Antirepeat sequence of processed tracrRNA (red) and repeat-derived sequence of mature crRNA (grey) were co-folded for each type II CRISPR-Cas locus studied (see Materials and Methods). Color bars indicated on the left group dual-RNAs from loci with closely related Cas9 (see FIG. 1 and Supplementary FIG. S4 ). RNA duplexes belonging to the same groups display structural similarities, suggesting a role of the structure in dual-RNA recognition by Cas9.
  • polynucleotide and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
  • Oligonucleotide generally refers to polynucleotides of between about 5 and about 100 nucleotides of single- or double-stranded DNA.
  • oligonucleotide is also known as “oligomers” or “oligos” and may be isolated from genes, or chemically synthesized by methods known in the art.
  • polynucleotide and nucleic acid should be understood to include, as applicable to the embodiments being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides.
  • Genomic DNA refers to the DNA of a genome of an organism including, but not limited to, the DNA of the genome of a bacterium, fungus, archea, plant or animal.
  • Manipulating DNA encompasses binding, nicking one strand, or cleaving (i.e., cutting) both strands of the DNA, or encompasses modifying the DNA or a polypeptide associated with the DNA (e.g., the modifications of paragraphs [00161] or [00162]).
  • Manipulating DNA can silence, activate, or modulate (either increase or decrease) the expression of an RNA or polypeptide encoded by the DNA.
  • a “stem-loop structure” refers to a nucleic acid having a secondary structure that includes a region of nucleotides which are known or predicted to form a double strand (stem portion) that is linked on one side by a region of predominantly single-stranded nucleotides (loop portion).
  • the terms “hairpin” and “fold-back” structures are also used herein to refer to stem-loop structures. Such structures are well known in the art and these terms are used consistently with their known meanings in the art.
  • a stem-loop structure does not require exact base-pairing.
  • the stem may include one or more base mismatches.
  • the base-pairing may be exact, i.e. not include any mismatches.
  • hybridizable or “complementary” or “substantially complementary” it is meant that a nucleic acid (e.g. RNA) comprises a sequence of nucleotides that enables it to non-covalently bind, i.e. form Watson-Crick base pairs and/or G/U base pairs, “anneal”, or “hybridize,” to another nucleic acid in a sequence-specific, antiparallel, manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid) under the appropriate in vitro and/or in vivo conditions of temperature and solution ionic strength.
  • RNA complementary nucleic acid
  • standard Watson-Crick base-pairing includes: adenine (A) pairing with thymidine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine (C) [DNA, RNA].
  • A adenine
  • U uracil
  • G guanine
  • C cytosine
  • G/U base-pairing is partially responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anti-codon base-pairing with codons in mRNA.
  • a guanine (G) of a protein-binding segment (dsRNA duplex) of a guide RNA molecule is considered complementary to a uracil (U), and vice versa.
  • G guanine
  • U uracil
  • Hybridization and washing conditions are well known and exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1 therein; and Sambrook, J. and Russell, W., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001).
  • the conditions of temperature and ionic strength determine the “stringency” of the hybridization.
  • Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases are possible.
  • the conditions appropriate for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of complementation between two nucleotide sequences, the greater the value of the melting temperature (Tm) for hybrids of nucleic acids having those sequences.
  • Tm melting temperature
  • For hybridizations between nucleic acids with short stretches of complementarity e.g. complementarity over 35 or less, 30 or less, 25 or less, 22 or less, 20 or less, or 18 or less nucleotides
  • the position of mismatches becomes important (see Sambrook et al., supra, 11.7-11.8).
  • the length for a hybridizable nucleic acid is at least about 10 nucleotides.
  • Illustrative minimum lengths for a hybridizable nucleic acid are: at least about 15 nucleotides; at least about 20 nucleotides; at least about 22 nucleotides; at least about 25 nucleotides; and at least about 30 nucleotides).
  • the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the region of complementation and the degree of complementation.
  • polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable or hybridizable. Moreover, a polynucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure).
  • a polynucleotide can comprise at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence complementarity to a target region within the target nucleic acid sequence to which they are targeted.
  • an antisense nucleic acid in which 18 of 20 nucleotides of the antisense compound are complementary to a target region, and would therefore specifically hybridize would represent 90 percent complementarity.
  • the remaining noncomplementary nucleotides may be clustered or interspersed with complementary nucleotides and need not be contiguous to each other or to complementary nucleotides. Percent complementarity between particular stretches of nucleic acid sequences within nucleic acids can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol.
  • peptide refers to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.
  • Binding refers to a non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid). While in a state of non-covalent interaction, the macromolecules are said to be “associated” or “interacting” or “binding” (e.g., when a molecule X is said to interact with a molecule Y, it is meant the molecule X binds to molecule Y in a non-covalent manner).
  • Binding interactions are generally characterized by a dissociation constant (K d ) of less than 10 ⁇ 6 M, less than 10 ⁇ 7 M, less than 10 ⁇ 8 M, less than 10 ⁇ 9 M, less than 10 ⁇ 10 M, less than 10 ⁇ 11 M, less than 10 ⁇ 12 M, less than 10 ⁇ 13 M, less than 10 ⁇ 14 M, or less than 10 ⁇ 15 M.
  • K d dissociation constant
  • Affinity refers to the strength of binding, increased binding affinity being correlated with a lower K d .
  • binding domain it is meant a protein domain that is able to bind non-covalently to another molecule.
  • a binding domain can bind to, for example, a DNA molecule (a DNA-binding protein), an RNA molecule (an RNA-binding protein) and/or a protein molecule (a protein-binding protein).
  • a protein domain-binding protein it can bind to itself (to form homodimers, homotrimers, etc.) and/or it can bind to one or more molecules of a different protein or proteins.
  • a group of amino acids having aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains consists of serine and threonine; a group of amino acids having amide containing side chains consisting of asparagine and glutamine; a group of amino acids having aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains consists of lysine, arginine, and histidine; a group of amino acids having acidic side chains consists of glutamate and aspartate; and a group of amino acids having sulfur containing side chains consists of cysteine and methionine.
  • Exemplary conservative amino acid substitution groups are: valine-leucine-isoleucine, phenyla
  • a polynucleotide or polypeptide has a certain percent “sequence identity” to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences. Sequence identity can be determined in a number of different manners.
  • sequences can be aligned using various methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.), available over the world wide web at sites including ncbi.nlm.nili.gov/BLAST, ebi.ac.uk/Tools/msa/tcoffee/, ebi.ac.uk/Tools/msa/muscle/, mafft.cbrc.jp/alignment/software/. See, e.g., Altschul et al. (1990), J. Mol. Bioi. 215:403-10.
  • Sequence alignments standard in the art are used according to the invention to determine amino acid residues in a Cas9 ortholog that “correspond to” amino acid residues in another Cas9 ortholog.
  • the amino acid residues of Cas9 orthologs that correspond to amino acid residues of other Cas9 orthologs appear at the same position in alignments of the sequences.
  • a DNA sequence that “encodes” a particular RNA is a DNA nucleic acid sequence that is transcribed into RNA.
  • a DNA polynucleotide may encode an RNA (mRNA) that is translated into protein, or a DNA polynucleotide may encode an RNA that is not translated into protein (e.g. tRNA, rRNA, or a guide RNA; also called “non-coding” RNA or “ncRNA”).
  • a “protein coding sequence or a sequence that encodes a particular protein or polypeptide is a nucleic acid sequence that is transcribed into mRNA (in the case of DNA) and is translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences.
  • the boundaries of the coding sequence are determined by a start codon at the 5′ terminus (N-terminus) and a translation stop nonsense codon at the 3′ terminus (C-terminus).
  • a coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic nucleic acids.
  • a transcription termination sequence will usually be located 3′ to the coding sequence.
  • a “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase and initiating transcription of a downstream (3′ direction) coding or non-coding sequence.
  • the promoter sequence is bounded at its 3′ terminus by the transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site within the promoter sequence will be found a transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase.
  • Eukaryotic promoters will often, but not always, contain “TATA” boxes and “CAT” boxes.
  • Various promoters, including inducible promoters may be used to drive the various vectors of the present invention.
  • a promoter can be a constitutively active promoter (i.e., a promoter that is constitutively in an active/“ON” state), it may be an inducible promoter (i.e., a promoter whose state, active/“ON” or inactive/“OFF”, is controlled by an external stimulus, e.g., the presence of a particular temperature, compound, or protein), it may be a spatially restricted promoter (i.e., transcriptional control element, enhancer, etc.)(e.g., tissue specific promoter, cell type specific promoter, etc.), and it may be a temporally restricted promoter (i.e., the promoter is in the “ON” state or “OFF” state during specific stages of embryonic development or during specific stages of a biological process, e.g., hair follicle cycle in mice).
  • a constitutively active promoter i.e., a promoter that is constitutively in an active/“ON” state
  • it may be an inducible promoter
  • Suitable promoters can be derived from viruses and can therefore be referred to as viral promoters, or they can be derived from any organism, including prokaryotic or eukaryotic organisms. Suitable promoters can be used to drive expression by any RNA polymerase (e.g., pol I, pol II, pol III).
  • RNA polymerase e.g., pol I, pol II, pol III
  • Exemplary promoters include, but are not limited to the SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, a human U6 small nuclear promoter (U6) (Miyagishi et al., Nature Biotechnology 20, 497-500 (2002)), an enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep. 1; 31(17)), a human H1 promoter (H1), and the like.
  • LTR mouse mammary tumor virus long terminal repeat
  • Ad MLP adenovirus major late promoter
  • HSV herpes simplex virus
  • CMV cytomegalovirus
  • CMVIE C
  • inducible promoters include, but are not limited to T7 RNA polymerase promoter, T3 RNA polymerase promoter, Isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated promoter, lactose induced promoter, heat shock promoter, Tetracycline-regulated promoter, Steroid-regulated promoter, Metal-regulated promoter, estrogen receptor-regulated promoter, etc.
  • Inducible promoters can therefore be regulated by molecules including, but not limited to, doxycycline; RNA polymerase, e.g., T7 RNA polymerase; an estrogen receptor; an estrogen receptor fusion; etc.
  • the promoter is a spatially restricted promoter (i.e., cell type specific promoter, tissue specific promoter, etc.) such that in a multi-cellular organism, the promoter is active (i.e., “ON”) in a subset of specific cells.
  • spatially restricted promoters may also be referred to as enhancers, transcriptional control elements, control sequences, etc.
  • any convenient spatially restricted promoter may be used and the choice of suitable promoter (e.g., a brain specific promoter, a promoter that drives expression in a subset of neurons, a promoter that drives expression in the germline, a promoter that drives expression in the lungs, a promoter that drives expression in muscles, a promoter that drives expression in islet cells of the pancreas, etc.) will depend on the organism.
  • various spatially restricted promoters are known for plants, flies, worms, mammals, mice, etc.
  • a spatially restricted promoter can be used to regulate the expression of a nucleic acid encoding a site-directed modifying polypeptide in a wide variety of different tissues and cell types, depending on the organism.
  • Some spatially restricted promoters are also temporally restricted such that the promoter is in the “ON” state or “OFF” state during specific stages of embryonic development or during specific stages of a biological process (e.g., hair follicle cycle in mice).
  • spatially restricted promoters include, but are not limited to, neuron-specific promoters, adipocyte-specific promoters, cardiomyocyte-specific promoters, smooth muscle-specific promoters, photoreceptor-specific promoters, etc.
  • Neuron-specific spatially restricted promoters include, but are not limited to, a neuron-specific enolase (NSE) promoter (see, e.g., EMBL HSENO2, X51956); an aromatic amino acid decarboxylase (AADC) promoter; a neurofilament promoter (see, e.g., GenBank HUMNFL, L04147); a synapsin promoter (see, e.g., GenBank HUMSYNIB, M55301); a thy-1 promoter (see, e.g., Chen et al. (1987) Cell 51:7-19; and Llewellyn, et al. (2010) Nat. Med.
  • NSE neuron-specific enolase
  • AADC aromatic amino acid decarboxylase
  • Adipocyte-specific spatially restricted promoters include, but are not limited to aP2 gene promoter/enhancer, e.g., a region from ⁇ 5.4 kb to +21 bp of a human aP2 gene (see, e.g., Tozzo et al. (1997) Endocrinol. 138:1604; Ross et al. (1990) Proc. Natl. Acad. Sci. USA 87:9590; and Pavjani et al. (2005) Nat. Med. 11:797); a glucose transporter-4 (GLUT4) promoter (see, e.g., Knight et al. (2003) Proc. Natl. Acad. Sci.
  • aP2 gene promoter/enhancer e.g., a region from ⁇ 5.4 kb to +21 bp of a human aP2 gene (see, e.g., Tozzo et al. (1997) Endocrinol. 138:160
  • fatty acid translocase (FAT/CD36) promoter see, e.g., Kuriki et al. (2002) Biol. Pharm. Bull. 25:1476; and Sato et al. (2002) J. Biol. Chem. 277:15703
  • SCD1 stearoyl-CoA desaturase-1
  • SCD1 stearoyl-CoA desaturase-1 promoter
  • leptin promoter see, e.g., Mason et al. (1998) Endocrinol. 139:1013; and Chen et al. (1999) Biochem. Biophys. Res. Comm.
  • adiponectin promoter see, e.g., Kita et al. (2005) Biochem. Biophys. Res. Comm. 331:484; and Chakrabarti (2010) Endocrinol. 151:2408
  • an adipsin promoter see, e.g., Platt et al. (1989) Proc. Natl. Acad. Sci. USA 86:7490
  • a resistin promoter see, e.g., Seo et al. (2003) Molec. Endocrinol. 17:1522); and the like.
  • Cardiomyocyte-specific spatially restricted promoters include, but are not limited to control sequences derived from the following genes: myosin light chain-2, a-myosin heavy chain, AE3, cardiac troponin C, cardiac actin, and the like.
  • Franz et al. (1997) Cardiovasc. Res. 35:560-566; Robbins et al. (1995) Ann. N.Y. Acad. Sci. 752:492-505; Linn et al. (1995) Circ. Res. 76:584591; Parmacek et al. (1994) Mol. Cell. Biol. 14:1870-1885; Hunter et al. (1993) Hypertension 22:608-617; and Sartorelli et al. (1992) Proc. Natl. Acad. Sci. USA 89:4047-4051.
  • Smooth muscle-specific spatially restricted promoters include, but are not limited to an SM22a promoter (see, e.g., Akyiirek et al. (2000) Mol. Med. 6:983; and U.S. Pat. No. 7,169,874); a smoothelin promoter (see, e.g., WO 2001/018048); an a-smooth muscle actin promoter; and the like.
  • a 0.4 kb region of the SM22a promoter, within which lie two CArG elements has been shown to mediate vascular smooth muscle cell-specific expression (see, e.g., Kim, et al. (1997) Mol. Cell. Biol. 17, 2266-2278; Li, et al., (1996) J. Cell Biol. 132, 849-859; and Moessler, et al. (1996) Development 122, 2415-2425).
  • Photoreceptor-specific spatially restricted promoters include, but are not limited to, a rhodopsin promoter; a rhodopsin kinase promoter (Young et al. (2003) Ophthalmol. Vis. Sci. 44:4076); a beta phosphodiesterase gene promoter (Nicoud et al. (2007) J. Gene Med. 9:1015); a retinitis pigmentosa gene promoter (Nicoud et al. (2007) supra); an interphotoreceptor retinoid-binding protein (IRBP) gene enhancer (Nicoud et al. (2007) supra); an IRBP gene promoter (Yokoyama et al. (1992) Exp Eye Res. 55:225); and the like.
  • a rhodopsin promoter a rhodopsin kinase promoter
  • a beta phosphodiesterase gene promoter Necoud et al. (2007) J. Gene
  • DNA regulatory sequences refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and/or regulate transcription of a non-coding sequence (e.g., guide RNA) or a coding sequence (e.g., site-directed modifying polypeptide, or Cas9 polypeptide) and/or regulate translation of an encoded polypeptide.
  • a non-coding sequence e.g., guide RNA
  • a coding sequence e.g., site-directed modifying polypeptide, or Cas9 polypeptide
  • nucleic acid refers to a nucleic acid, polypeptide, cell, or organism that is found in nature.
  • a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by a human in the laboratory is naturally occurring.
  • chimeric refers to two components that are defined by structures derived from different sources.
  • a chimeric polypeptide e.g., a chimeric Cas9 protein
  • the chimeric polypeptide includes amino acid sequences that are derived from different polypeptides.
  • a chimeric polypeptide may comprise either modified or naturally-occurring polypeptide sequences (e.g., a first amino acid sequence from a modified or unmodified Cas9 protein; and a second amino acid sequence other than the Cas9 protein).
  • chimeric in the context of a polynucleotide encoding a chimeric polypeptide includes nucleotide sequences derived from different coding regions (e.g., a first nucleotide sequence encoding a modified or unmodified Cas9 protein; and a second nucleotide sequence encoding a polypeptide other than a Cas9 protein).
  • chimeric polypeptide refers to a polypeptide which is not naturally occurring, e.g., is made by the artificial combination (i.e., “fusion”) of two otherwise separated segments of amino sequence through human intervention.
  • a polypeptide that comprises a chimeric amino acid sequence is a chimeric polypeptide.
  • Some chimeric polypeptides can be referred to as “fusion variants.”
  • Heterologous means a nucleotide or peptide that is not found in the native nucleic acid or protein, respectively.
  • the RNA-binding domain of a naturally-occurring bacterial Cas9 polypeptide may be fused to a heterologous polypeptide sequence (i.e. a polypeptide sequence from a protein other than Cas9 or a polypeptide sequence from another organism).
  • the heterologous polypeptide may exhibit an activity (e.g., enzymatic activity) that will also be exhibited by the chimeric Cas9 protein (e.g., methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitinating activity, etc.).
  • a heterologous nucleic acid may be linked to a naturally-occurring nucleic acid (or a variant thereof) (e.g., by genetic engineering) to generate a chimeric polynucleotide encoding a chimeric polypeptide.
  • a variant Cas9 site-directed polypeptide may be fused to a heterologous polypeptide (i.e. a polypeptide other than Cas9), which exhibits an activity that will also be exhibited by the fusion variant Cas9 site-directed polypeptide.
  • a heterologous nucleic acid may be linked to a variant Cas9 site-directed polypeptide (e.g., by genetic engineering) to generate a polynucleotide encoding a fusion variant Cas9 site-directed polypeptide.
  • “Heterologous,” as used herein, additionally means a nucleotide or polypeptide in a cell that is not its native cell.
  • cognate refers to two biomolecules that normally interact or co-exist in nature.
  • Recombinant means that a particular nucleic acid (DNA or RNA) or vector is the product of various combinations of cloning, restriction, polymerase chain reaction (PCR) and/or ligation steps resulting in a construct having a structural coding or non-coding sequence distinguishable from endogenous nucleic acids found in natural systems.
  • DNA sequences encoding polypeptides can be assembled from cDNA fragments or from a series of synthetic oligonucleotides, to provide a synthetic nucleic acid which is capable of being expressed from a recombinant transcriptional unit contained in a cell or in a cell-free transcription and translation system.
  • Genomic DNA comprising the relevant sequences can also be used in the formation of a recombinant gene or transcriptional unit. Sequences of non-translated DNA may be present 5′ or 3′ from the open reading frame, where such sequences do not interfere with manipulation or expression of the coding regions, and may indeed act to modulate production of a desired product by various mechanisms (see “DNA regulatory sequences”, below). Alternatively, DNA sequences encoding RNA (e.g., guide RNA) that is not translated may also be considered recombinant. Thus, e.g., the term “recombinant” nucleic acid refers to one which is not naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of sequence through human intervention.
  • This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. Such is usually done to replace a codon with a codon encoding the same amino acid, a conservative amino acid, or a non-conservative amino acid. Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a desired combination of functions. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.
  • a recombinant polynucleotide encodes a polypeptide
  • the sequence of the encoded polypeptide can be naturally occurring (“wild type”) or can be a variant (e.g., a mutant) of the naturally occurring sequence.
  • the term “recombinant” polypeptide does not necessarily refer to a polypeptide whose sequence does not naturally occur.
  • a “recombinant” polypeptide is encoded by a recombinant DNA sequence, but the sequence of the polypeptide can be naturally occurring (“wild type”) or non-naturally occurring (e.g., a variant, a mutant, etc.).
  • a “recombinant” polypeptide is the result of human intervention, but may be a naturally occurring amino acid sequence.
  • a “vector” or “expression vector” is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, i.e. an “insert”, may be attached so as to bring about the replication of the attached segment in a cell.
  • an “expression cassette” comprises a DNA coding sequence operably linked to a promoter.
  • “Operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression.
  • the terms “recombinant expression vector,” or “DNA construct” are used interchangeably herein to refer to a DNA molecule comprising a vector and at least one insert. Recombinant expression vectors are usually generated for the purpose of expressing and/or propagating the insert(s), or for the construction of other recombinant nucleotide sequences.
  • the nucleic acid(s) may or may not be operably linked to a promoter sequence and may or may not be operably linked to DNA regulatory sequences.
  • a cell has been “genetically modified” or “transformed” or “transfected” by exogenous DNA, e.g. a recombinant expression vector, when such DNA has been introduced inside the cell.
  • exogenous DNA e.g. a recombinant expression vector
  • the presence of the exogenous DNA results in permanent or transient genetic change.
  • the transforming DNA may or may not be integrated (covalently linked) into the genome of the cell.
  • the transforming DNA may be maintained on an episomal element such as a plasmid.
  • a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones that comprise a population of daughter cells containing the transforming DNA.
  • a “clone” is a population of cells derived from a single cell or common ancestor by mitosis.
  • a “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
  • Suitable methods of genetic modification include e.g., viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, nanoparticle-mediated nucleic acid delivery (see, e.g., Panyam et., al Adv Drug Deliv Rev. 2012 Sep. 13. pii: 50169-409X(12)00283-9. doi: 10.1016/j.addr.2012.09.023), and the like.
  • transformation include e.g., viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology,
  • a “host cell,” as used herein, denotes an in vivo or in vitro eukaryotic cell, a prokaryotic cell (e.g., bacterial or archaeal cell), or a cell from a multicellular organism (e.g., a cell line) cultured as a unicellular entity, which eukaryotic or prokaryotic cells can be, or have been, used as recipients for a nucleic acid, and include the progeny of the original cell which has been transformed by the nucleic acid. It is understood that the progeny of a single cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation.
  • a “recombinant host cell” is a host cell into which has been introduced a heterologous nucleic acid, e.g., an expression vector.
  • a bacterial host cell is a genetically modified bacterial host cell by virtue of introduction into a suitable bacterial host cell of an exogenous nucleic acid (e.g., a plasmid or recombinant expression vector) and a eukaryotic host cell is a genetically modified eukaryotic host cell (e.g., a mammalian germ cell), by virtue of introduction into a suitable eukaryotic host cell of an exogenous nucleic acid.
  • a “target DNA” as used herein is a DNA polynucleotide that comprises a “target site” or “target sequence.”
  • target site a DNA polynucleotide that comprises a “target site” or “target sequence.”
  • target site a DNA polynucleotide that comprises a “target site” or “target sequence.”
  • target sequence a DNA polynucleotide that comprises a “target site” or “target sequence.”
  • target site target sequence
  • target protospacer DNA or “protospacer-like sequence” are used interchangeably herein to refer to a nucleic acid sequence present in a target DNA to which a DNA-targeting segment of a guide RNA will bind, provided sufficient conditions for binding exist.
  • the target site (or target sequence) 5′-GAGCATATC-3′ within a target DNA is targeted by (or is bound by, or hybridizes with, or is complementary to) the RNA sequence 5′-GAUAUGCUC-3′.
  • Suitable DNA/RNA binding conditions include
  • RNA/RNA binding conditions e.g., conditions in a cell-free system
  • the strand of the target DNA that is complementary to and hybridizes with the guide RNA is referred to as the “complementary strand”
  • the strand of the target DNA that is complementary to the “complementary strand” (and is therefore not complementary to the guide RNA) is referred to as the “noncomplementary strand” or “non-complementary strand.”
  • site-directed modifying polypeptide” or “RNA-binding site-directed polypeptide” or “RNA-binding site-directed modifying polypeptide” or “site-directed polypeptide” it is meant a polypeptide that binds RNA and is targeted to a specific DNA sequence.
  • a site-directed modifying polypeptide as described herein is targeted to a specific DNA sequence by the RNA molecule to which it is bound.
  • the RNA molecule comprises a sequence that binds, hybridizes to, or is complementary to a target sequence within the target DNA, thus targeting the bound polypeptide to a specific location within the target DNA (the target sequence).
  • Exemplary target sequences of the invention are set out in SEQ ID NOs: 801-2701.
  • SEQ ID NOs: 801-973 are protospacer-like target sequences 5′ to the PAM sequence NNNNACA in the human CCR5 gene.
  • SEQ ID NOs: 974-1078 are protospacer-like sequences 5′ to the PAM sequence GNNNCNNA in the human CCR5 gene.
  • SEQ ID NOs: 1079-1222 are protospacer-like target sequences 5′ to the PAM sequence NNNNACA in the exons of the human CCR5 gene.
  • SEQ ID NOs: 1223-1312 are protospacer-like sequences 5′ to the PAM sequence GNNNCNNA in the exons of the human CCR5 gene.
  • SEQ ID NOs: 1313-1348 are protospacer-like target sequences 5′ to the PAM sequence NNNNACA around the 5′ end of the human CCR5 gene.
  • SEQ ID NOs: 1349-1371 are protospacer-like sequences 5′ to the PAM sequence GNNNCNNA around the 5′ end of the human CCR5 gene.
  • SEQ ID NOs: 1372-1415 are protospacer-like target sequences 5′ to the PAM sequence NNNNACA around the delta 32 locus in the human CCR5 gene.
  • SEQ ID NOs: 1416-1443 are protospacer-like sequences 5′ to the PAM sequence GNNNCNNA around the delta 32 locus in the human CCR5 gene.
  • SEQ ID NOs: 1444-1900 are protospacer-like target sequences 5′ to the PAM sequence NNNNACA in the human BCL11A gene.
  • SEQ ID NOs: 1901-2162 are protospacer-like sequences 5′ to the PAM sequence GNNNCNNA in the human BCL11A gene.
  • SEQ ID NOs: 2163-2482 are protospacer-like target sequences 5′ to the PAM sequence NNNNACA in the exons of the human BCL11A gene.
  • SEQ ID NOs: 2483-2666 are protospacer-like sequences 5′ to the PAM sequence GNNNCNNA in the exons of the human BCL11A gene.
  • SEQ ID NOs: 2667-2686 are protospacer-like target sequences 5′ to the PAM sequence NNNNACA around the 5′ end of the human BCL11A gene.
  • SEQ ID NOs: 2687-2701 are protospacer-like sequences 5′ to the PAM sequence GNNNCNNA around the 5′ end of the human BCL11A gene.
  • Target sequences at least 80% identical to the sequences set out in SEQ ID NOs: 801-2701 are also contemplated.
  • cleavage it is meant the breakage of the covalent backbone of a DNA molecule. Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Both single-stranded cleavage and double-stranded cleavage are possible, and double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events. DNA cleavage can result in the production of either blunt ends or staggered ends.
  • a complex comprising a guide RNA and a site-directed modifying polypeptide is used for targeted double-stranded DNA cleavage.
  • Nuclease and “endonuclease” are used interchangeably herein to mean an enzyme which possesses endonucleolytic catalytic activity for DNA cleavage.
  • cleavage domain or “active domain” or “nuclease domain” of a nuclease it is meant the polypeptide sequence or domain within the nuclease which possesses the catalytic activity for DNA cleavage.
  • a cleavage domain can be contained in a single polypeptide chain or cleavage activity can result from the association of two (or more) polypeptides.
  • a single nuclease domain may consist of more than one isolated stretch of amino acids within a given polypeptide.
  • site-directed polypeptide or “RNA-binding site-directed polypeptide” or “RNA-binding site-directed polypeptide” it is meant a polypeptide that binds RNA and is targeted to a specific DNA sequence.
  • a site-directed polypeptide as described herein is targeted to a specific DNA sequence by the RNA molecule to which it is bound.
  • the RNA molecule comprises a sequence that is complementary to a target sequence within the target DNA, thus targeting the bound polypeptide to a specific location within the target DNA (the target sequence).
  • RNA molecule that binds to the site-directed modifying polypeptide and targets the polypeptide to a specific location within the target DNA is referred to herein as the “guide RNA” or “guide RNA polynucleotide” (also referred to herein as a “guide RNA” or “gRNA”).
  • a guide RNA comprises two segments, a “DNA-targeting segment” and a “protein-binding segment.”
  • segment it is meant a segment/section/region of a molecule, e.g., a contiguous stretch of nucleotides in an RNA.
  • a segment can also mean a region/section of a complex such that a segment may comprise regions of more than one molecule.
  • the protein-binding segment (described below) of a guide RNA is one RNA molecule and the protein-binding segment therefore comprises a region of that RNA molecule.
  • the protein-binding segment (described below) of a guide RNA comprises two separate molecules that are hybridized along a region of complementarity.
  • a protein-binding segment of a guide RNA that comprises two separate molecules can comprise (i) base pairs 40-75 of a first RNA molecule that is 100 base pairs in length; and (ii) base pairs 10-25 of a second RNA molecule that is 50 base pairs in length.
  • segment unless otherwise specifically defined in a particular context, is not limited to a specific number of total base pairs, is not limited to any particular number of base pairs from a given RNA molecule, is not limited to a particular number of separate molecules within a complex, and may include regions of RNA molecules that are of any total length and may or may not include regions with complementarity to other molecules.
  • the DNA-targeting segment (or “DNA-targeting sequence”) comprises a nucleotide sequence that is complementary to a specific sequence within a target DNA (the complementary strand of the target DNA) designated the “protospacer-like” sequence herein.
  • the protein-binding segment (or “protein-binding sequence”) interacts with a site-directed modifying polypeptide.
  • site-directed modifying polypeptide is a Cas9 or Cas9 related polypeptide (described in more detail below)
  • site-specific cleavage of the target DNA occurs at locations determined by both (i) base-pairing complementarity between the guide RNA and the target DNA; and (ii) a short motif (referred to as the protospacer adjacent motif (PAM)) in the target DNA.
  • PAM protospacer adjacent motif
  • the protein-binding segment of a guide RNA comprises, in part, two complementary stretches of nucleotides that hybridize to one another to form a double stranded RNA duplex (dsRNA duplex).
  • a nucleic acid (e.g., a guide RNA, a nucleic acid comprising a nucleotide sequence encoding a guide RNA; a nucleic acid encoding a site-directed polypeptide; etc.) comprises a modification or sequence that provides for an additional desirable feature (e.g., modified or regulated stability; subcellular targeting; tracking, e.g., a fluorescent label; a binding site for a protein or protein complex; etc.).
  • an additional desirable feature e.g., modified or regulated stability; subcellular targeting; tracking, e.g., a fluorescent label; a binding site for a protein or protein complex; etc.
  • Non-limiting examples include: a 5′ cap (e.g., a 7-methylguanylate cap (m7G)); a 3′ polyadenylated tail (i.e., a 3′ poly(A) tail); a riboswitch sequence (e.g., to allow for regulated stability and/or regulated accessibility by proteins and/or protein complexes); a stability control sequence; a sequence that forms a dsRNA duplex (i.e., a hairpin)); a modification or sequence that targets the RNA to a subcellular location (e.g., nucleus, mitochondria, chloroplasts, and the like); a modification or sequence that provides for tracking (e.g., direct conjugation to a fluorescent molecule, conjugation to a moiety that facilitates fluorescent detection, a sequence that allows for fluorescent detection, etc.); a modification or sequence that provides a binding site for proteins (e.g., proteins that act on DNA, including transcriptional activators, transcriptional repressors, DNA
  • a guide RNA comprises an additional segment at either the 5′ or 3′ end that provides for any of the features described above.
  • a suitable third segment can comprise a 5′ cap (e.g., a 7-methylguanylate cap (m7G)); a 3′ polyadenylated tail (i.e., a 3′ poly(A) tail); a riboswitch sequence (e.g., to allow for regulated stability and/or regulated accessibility by proteins and protein complexes); a stability control sequence; a sequence that forms a dsRNA duplex (i.e., a hairpin)); a sequence that targets the RNA to a subcellular location (e.g., nucleus, mitochondria, chloroplasts, and the like); a modification or sequence that provides for tracking (e.g., direct conjugation to a fluorescent molecule, conjugation to a moiety that facilitates fluorescent detection, a sequence that allows for fluorescent detection, etc.); a modification or sequence that provides for tracking (e.g
  • a guide RNA and a site-directed modifying polypeptide form a complex (i.e., bind via non-covalent interactions).
  • the guide RNA provides target specificity to the complex by comprising a nucleotide sequence that is complementary to a sequence of a target DNA.
  • the site-directed modifying polypeptide of the complex provides the site-specific activity.
  • the site-directed modifying polypeptide is guided to a target DNA sequence (e.g. a target sequence in a chromosomal nucleic acid; a target sequence in an extrachromosomal nucleic acid, e.g.
  • a guide RNA comprises two separate RNA molecules (RNA polynucleotides: an “activator-RNA” and a “targeter-RNA”, see below) and is referred to herein as a “double-molecule guide RNA” or a “two-molecule guide RNA.”
  • the guide RNA is a single RNA molecule (single RNA polynucleotide) and is referred to herein as a “single-molecule guide RNA,” a “single-guide RNA,” or an “sgRNA.”
  • the term “guide RNA” or “gRNA” is inclusive, referring both to double-molecule guide RNAs and to single-molecule guide RNAs (i.e., sgRNAs).
  • a two-molecule guide RNA comprises two separate RNA molecules (a “targeter-RNA” and an “activator-RNA”).
  • Each of the two RNA molecules of a two-molecule guide RNA comprises a stretch of nucleotides that are complementary to one another such that the complementary nucleotides of the two RNA molecules hybridize to form the double stranded RNA duplex of the protein-binding segment.
  • An exemplary two-molecule guide RNA comprises a crRNA-like (“CRISPR RNA” or “targeter-RNA”) molecule (which includes a CRISPR repeat or CRISPR repeat-like sequence) and a corresponding tracrRNA-like (“trans-activating CRISPR RNA” or “activator-RNA” or “tracrRNA”) molecule.
  • CRISPR RNA or “targeter-RNA”
  • targeter-RNA comprises both the DNA-targeting segment (single stranded) of the guide RNA and a stretch (“duplex-forming segment”) of nucleotides that forms one half of the dsRNA duplex of the protein-binding segment of the guide RNA.
  • a corresponding tracrRNA-like molecule comprises a stretch of nucleotides (duplex-forming segment) that forms the other half of the dsRNA duplex of the protein-binding segment of the guide RNA.
  • a stretch of nucleotides of a crRNA-like molecule are complementary to and hybridize with a stretch of nucleotides of a tracrRNA-like molecule to form the dsRNA duplex of the protein-binding domain of the guide RNA.
  • each crRNA-like molecule can be said to have a corresponding tracrRNA-like molecule.
  • the crRNA-like molecule additionally provides the single stranded DNA-targeting segment.
  • a crRNA-like and a tracrRNA-like molecule hybridize to form a guide RNA.
  • a double-molecule guide RNA can comprise any corresponding crRNA and tracrRNA pair.
  • a two-molecule guide RNA can be designed to allow for controlled (i.e., conditional) binding of a targeter-RNA with an activator-RNA. Because a two-molecule guide RNA is not functional unless both the activator-RNA and the targeter-RNA are bound in a functional complex with Cas9, a two-molecule guide RNA can be inducible (e.g., drug inducible) by rendering the binding between the activator-RNA and the targeter-RNA to be inducible.
  • RNA aptamers can be used to regulate (i.e., control) the binding of the activator-RNA with the targeter-RNA. Accordingly, the activator-RNA and/or the targeter-RNA can comprise an RNA aptamer sequence.
  • a single-molecule guide RNA comprises two stretches of nucleotides (a targeter-RNA and an activator-RNA) that are complementary to one another, are covalently linked (directly, or by intervening nucleotides), and hybridize to form the double stranded RNA duplex (dsRNA duplex) of the protein-binding segment, thus resulting in a stem-loop structure.
  • the targeter-RNA and the activator-RNA can be covalently linked via the 3′ end of the targeter-RNA and the 5′ end of the activator-RNA.
  • targeter-RNA and the activator-RNA can be covalently linked via the 5′ end of the targeter-RNA and the 3′ end of the activator-RNA.
  • An exemplary single-molecule guide RNA comprises two complementary stretches of nucleotides that hybridize to form a dsRNA duplex.
  • one of the two complementary stretches of nucleotides of the single-molecule guide RNA (or the DNA encoding the stretch) is at least about 60% Identical to one of the activator-RNA (tracrRNA) sequences set forth in Supplementary Table S5 over a stretch of at least 8 contiguous nucleotides.
  • one of the two complementary stretches of nucleotides of the single-molecule guide RNA is at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical or 100% identical to one of the tracrRNA sequences set forth in Supplementary Table S5 over a stretch of at least 8 contiguous, at least 9 contiguous, at least 10 contiguous, at least 11 contiguous, at least 12 contiguous, at least 13 contiguous, at least 14 contiguous or at least 15 contiguous nucleotides.
  • the single-molecule guide RNA may comprise a nucleotide sequence that is at least 70% identical over at least 10 contiguous nucleotides, at least 80% identical over at least 10 contiguous nucleotides, at least 70% identical over at least 11 contiguous nucleotides, at least 80% identical over at least 11 contiguous nucleotides, at least 70% identical over at least 12 contiguous nucleotides, or at least 80% identical over at least 12 contiguous nucleotides of one of the tracrRNA sequences set forth in Supplementary Table S5.
  • one of the two complementary stretches of nucleotides of the single-molecule guide RNA is at least about 60% identical to one of the targeter-RNA (crRNA/CRISPR repeat) sequences set forth in Supplementary Table S5 over a stretch of at least 8 contiguous nucleotides.
  • one of the two complementary stretches of nucleotides of the single-molecule guide RNA is at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical or 100% identical to one of the crRNA/CRISPR repeat sequences set forth in Supplementary Table S5 over a stretch of at least 8 contiguous, at least 9 contiguous, at least 10 contiguous, at least 11 contiguous, at least 12 contiguous, at least 13 contiguous, at least 14 contiguous or at least 15 contiguous nucleotides.
  • the single-molecule guide RNA may comprise a nucleotide sequence that is at least 70% identical over at least 10 contiguous nucleotides, at least 80% identical over at least 10 contiguous nucleotides, at least 70% identical over at least 11 contiguous nucleotides, at least 80% identical over at least 11 contiguous nucleotides, at least 70% identical over at least 12 contiguous nucleotides, or at least 80% identical over at least 12 contiguous nucleotides of one of the CRISPR repeat sequences set forth in Supplementary Table S5.
  • activator-RNA is used herein to mean a tracrRNA-like molecule of a double-molecule guide RNA.
  • targeter-RNA is used herein to mean a crRNA-like molecule of a double-molecule guide RNA.
  • duplex-forming segment is used herein to mean the stretch of nucleotides of an activator-RNA or a targeter-RNA that contributes to the formation of the dsRNA duplex by hybridizing to a stretch of nucleotides of a corresponding activator-RNA or targeter-RNA molecule.
  • an activator-RNA comprises a duplex-forming segment that is complementary to the duplex-forming segment of the corresponding targeter-RNA.
  • an activator-RNA comprises a duplex-forming segment while a targeter-RNA comprises both a duplex-forming segment and the DNA-targeting segment of the guide RNA. Therefore, a double-molecule guide RNA can be comprised of any corresponding activator-RNA and targeter-RNA pair.
  • RNA aptamers are known in the art and are generally a synthetic version of a riboswitch.
  • the terms “RNA aptamer” and “riboswitch” are used interchangeably herein to encompass both synthetic and natural nucleic acid sequences that provide for inducible regulation of the structure (and therefore the availability of specific sequences) of the RNA molecule of which they are part.
  • RNA aptamers usually comprise a sequence that folds into a particular structure (e.g., a hairpin), which specifically binds a particular drug (e.g., a small molecule). Binding of the drug causes a structural change in the folding of the RNA, which changes a feature of the nucleic acid of which the aptamer is a part.
  • an activator-RNA with an aptamer may not be able to bind to the cognate targeter-RNA unless the aptamer is bound by the appropriate drug;
  • a targeter-RNA with an aptamer may not be able to bind to the cognate activator-RNA unless the aptamer is bound by the appropriate drug;
  • a targeter-RNA and an activator-RNA, each comprising a different aptamer that binds a different drug may not be able to bind to each other unless both drugs are present.
  • a two-molecule guide RNA can be designed to be inducible.
  • aptamers and riboswitches can be found, for example, in: Nakamura et al., Genes Cells. 2012 May; 17(5):344-64; Vavalle et al., Future Cardiol. 2012 May; 8(3):371-82; Citartan et al., Biosens Bioelectron. 2012 April 15; 34(1):1-11; and Liberman et al., Wiley Interdiscip Rev RNA. 2012 May-June; 3(3):369-84; all of which are herein incorporated by reference in their entirety.
  • stem cell is used herein to refer to a cell (e.g., plant stem cell, vertebrate stem cell) that has the ability both to self-renew and to generate a differentiated cell type (see Morrison et al. (1997) Cell 88:287-298).
  • the adjective “differentiated”, or “differentiating” is a relative term.
  • a “differentiated cell” is a cell that has progressed further down the developmental pathway than the cell it is being compared with.
  • pluripotent stem cells can differentiate into lineage-restricted progenitor cells (e.g., mesodermal stem cells), which in turn can differentiate into cells that are further restricted (e.g., neuron progenitors), which can differentiate into end-stage cells (i.e., terminally differentiated cells, e.g., neurons, cardiomyocytes, etc.), which play a characteristic role in a certain tissue type, and may or may not retain the capacity to proliferate further.
  • progenitor cells e.g., mesodermal stem cells
  • end-stage cells i.e., terminally differentiated cells, e.g., neurons, cardiomyocytes, etc.
  • Stem cells may be characterized by both the presence of specific markers (e.g., proteins, RNAs, etc.) and the absence of specific markers.
  • Stem cells may also be identified by functional assays both in vitro and in vivo, particularly assays relating to the ability of stem cells to give rise to multiple differentiated
  • PSCs pluripotent stem cells
  • Pluripotent stem cell or “PSC” is used herein to mean a stem cell capable of producing all cell types of the organism. Therefore, a PSC can give rise to cells of all germ layers of the organism (e.g., the endoderm, mesoderm, and ectoderm of a vertebrate). Pluripotent cells are capable of forming teratomas and of contributing to ectoderm, mesoderm, or endoderm tissues in a living organism. Pluripotent stem cells of plants are capable of giving rise to all cell types of the plant (e.g., cells of the root, stem, leaves, etc.).
  • PSCs of animals can be derived in a number of different ways.
  • ESCs embryonic stem cells
  • iPSCs induced pluripotent stem cells
  • somatic cells Takahashi et. al, Cell. 2007 November 30; 131(5):861-72; Takahashi et. al, Nat Protoc. 2007; 2(12):3081-9; Yu et. al, Science. 2007 December 21; 318(5858):1917-20. Epub 2007 November 20).
  • PSC refers to pluripotent stem cells regardless of their derivation
  • the term PSC encompasses the terms ESC and iPSC, as well as the term embryonic germ stem cells (EGSC), which are another example of a PSC.
  • ESC and iPSC as well as the term embryonic germ stem cells (EGSC), which are another example of a PSC.
  • EGSC embryonic germ stem cells
  • PSCs may be in the form of an established cell line, they may be obtained directly from primary embryonic tissue, or they may be derived from a somatic cell. PSCs can be target cells of the methods described herein.
  • ESC embryonic stem cell
  • ESC lines are listed in the NIH Human Embryonic Stem Cell Registry, e.g.
  • Stem cells of interest also include embryonic stem cells from other primates, such as Rhesus stem cells and marmoset stem cells.
  • the stem cells may be obtained from any mammalian species, e.g.
  • ESCs typically grow as flat colonies with large nucleo-cytoplasmic ratios, defined borders and prominent nucleoli.
  • ESCs express SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and Alkaline Phosphatase, but not SSEA-1.
  • Examples of methods of generating and characterizing ESCs may be found in, for example, U.S. Pat. No. 7,029,913, U.S. Pat. No. 5,843,780, and U.S. Pat. No. 6,200,806, the disclosures of which are incorporated herein by reference.
  • Methods for proliferating hESCs in the undifferentiated form are described in WO 99/20741, WO 01/51616, and WO 03/020920.
  • EGSC embryonic germ stem cell
  • EG cell a PSC that is derived from germ cells and/or germ cell progenitors, e.g. primordial germ cells, i.e. those that would become sperm and eggs.
  • Embryonic germ cells EG cells
  • Examples of methods of generating and characterizing EG cells may be found in, for example, U.S. Pat. No. 7,153,684; Matsui, Y., et al., (1992) Cell 70:841; Shamblott, M., et al. (2001) Proc. Natl. Acad. Sci.
  • iPSC induced pluripotent stem cell
  • iPSCs can be derived from multiple different cell types, including terminally differentiated cells. iPSCs have an ES cell-like morphology, growing as flat colonies with large nucleo-cytoplasmic ratios, defined borders and prominent nuclei.
  • iPSCs express one or more key pluripotency markers known by one of ordinary skill in the art, including but not limited to Alkaline Phosphatase, SSEA3, SSEA4, Sox2, Oct3/4, Nanog, TRA160, TRA181, TDGF 1, Dnmt3b, FoxD3, GDF3, Cyp26al, TERT, and zfp42.
  • Examples of methods of generating and characterizing iPSCs may be found in, for example, U.S. Patent Publication Nos. US20090047263, US20090068742, US20090191159, US20090227032, US20090246875, and US20090304646, the disclosures of which are incorporated herein by reference.
  • somatic cells are provided with reprogramming factors (e.g. Oct4, SOX2, KLF4, MYC, Nanog, Lin28, etc.) known in the art to reprogram the somatic cells to become pluripotent stem cells.
  • reprogramming factors e.g. Oct4, SOX2, KLF4, MYC, Nanog, Lin28, etc.
  • somatic cell it is meant any cell in an organism that, in the absence of experimental manipulation, does not ordinarily give rise to all types of cells in an organism.
  • somatic cells are cells that have differentiated sufficiently that they will not naturally generate cells of all three germ layers of the body, i.e. ectoderm, mesoderm and endoderm.
  • somatic cells would include both neurons and neural progenitors, the latter of which may be able to naturally give rise to all or some cell types of the central nervous system but cannot give rise to cells of the mesoderm or endoderm lineages.
  • mitotic cell it is meant a cell undergoing mitosis.
  • Mitosis is the process by which a eukaryotic cell separates the chromosomes in its nucleus into two identical sets in two separate nuclei. It is generally followed immediately by cytokinesis, which divides the nuclei, cytoplasm, organelles and cell membrane into two cells containing roughly equal shares of these cellular components.
  • post-mitotic cell it is meant a cell that has exited from mitosis, i.e., it is “quiescent”, i.e. it is no longer undergoing divisions. This quiescent state may be temporary, i.e. reversible, or it may be permanent.
  • meiotic cell it is meant a cell that is undergoing meiosis.
  • Meiosis is the process by which a cell divides its nuclear material for the purpose of producing gametes or spores. Unlike mitosis, in meiosis, the chromosomes undergo a recombination step which shuffles genetic material between chromosomes. Additionally, the outcome of meiosis is four (genetically unique) haploid cells, as compared with the two (genetically identical) diploid cells produced from mitosis.
  • HDR homology-directed repair
  • Homology-directed repair may result in an alteration of the sequence of the target molecule (e.g., insertion, deletion, mutation), if the donor polynucleotide differs from the target molecule and part or all of the sequence of the donor polynucleotide is incorporated into the target DNA.
  • the donor polynucleotide, a portion of the donor polynucleotide, a copy of the donor polynucleotide, or a portion of a copy of the donor polynucleotide integrates into the target DNA.
  • non-homologous end joining it is meant the repair of double-strand breaks in DNA by direct ligation of the break ends to one another without the need for a homologous template (in contrast to homology-directed repair, which requires a homologous sequence to guide repair). NHEJ often results in the loss (deletion) of nucleotide sequence near the site of the double-strand break.
  • treatment generally mean obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease or symptom in a mammal, and includes: (a) preventing the disease or symptom from occurring in a subject which may be predisposed to acquiring the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease or symptom, i.e., arresting its development; or (c) relieving the disease, i.e., causing regression of the disease.
  • the therapeutic agent may be administered before, during or after the onset of disease or injury.
  • the treatment of ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is of particular interest. Such treatment is desirably performed prior to complete loss of function in the affected tissues.
  • the therapy will desirably be administered during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease.
  • the terms “individual,” “subject,” “host,” and “patient,” are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans.
  • a guide RNA that directs the activities of an associated polypeptide (e.g., a site-directed modifying polypeptide) to a specific target sequence within a target DNA.
  • a guide RNA comprises: a first segment (also referred to herein as a “DNA-targeting segment” or a “DNA-targeting sequence”) and a second segment (also referred to herein as a “protein-binding segment” or a “protein-binding sequence”).
  • the DNA-targeting segment of a guide RNA comprises a nucleotide sequence that is complementary to a sequence in a target DNA.
  • the DNA-targeting segment of a guide RNA interacts with a target DNA in a sequence-specific manner via hybridization (i.e., base pairing).
  • the nucleotide sequence of the DNA-targeting segment may vary and determines the location within the target DNA that the guide RNA and the target DNA will interact.
  • the DNA-targeting segment of a guide RNA can be modified (e.g., by genetic engineering) to hybridize to any desired sequence within a target DNA.
  • the DNA-targeting segment can have a length of from about 12 nucleotides to about 100 nucleotides.
  • the DNA-targeting segment can have a length of from about 12 nucleotides (nt) to about 80 nt, from about 12 nt to about 50 nt, from about 12 nt to about 40 nt, from about 12 nt to about 30 nt, from about 12 nt to about 25 nt, from about 12 nt to about 20 nt, or from about 12 nt to about 19 nt.
  • the DNA-targeting segment can have a length of from about 19 nt to about 20 nt, from about 19 nt to about 25 nt, from about 19 nt to about 30 nt, from about 19 nt to about 35 nt, from about 19 nt to about 40 nt, from about 19 nt to about 45 nt, from about 19 nt to about 50 nt, from about 19 nt to about 60 nt, from about 19 nt to about 70 nt, from about 19 nt to about 80 nt, from about 19 nt to about 90 nt, from about 19 nt to about 100 nt, from about 20 nt to about 25 nt, from about 20 nt to about 30 nt, from about 20 nt to about 35 nt, from about 20 nt to about 40 nt, from about 20 nt to about 45 nt, from about 20 nt to about 50 nt, from about 20 nt,
  • the nucleotide sequence (the DNA-targeting sequence) of the DNA-targeting segment that is complementary to a nucleotide sequence (target sequence) of the target DNA can have a length at least about 12 nt.
  • the DNA-targeting sequence of the DNA-targeting segment that is complementary to a target sequence of the target DNA can have a length at least about 12 nt, at least about 15 nt, at least about 18 nt, at least about 19 nt, at least about 20 nt, at least about 25 nt, at least about 30 nt, at least about 35 nt or at least about 40 nt.
  • the DNA-targeting sequence of the DNA-targeting segment that is complementary to a target sequence of the target DNA can have a length of from about 12 nucleotides (nt) to about 80 nt, from about 12 nt to about 50 nt, from about 12 nt to about 45 nt, from about 12 nt to about 40 nt, from about 12 nt to about 35 nt, from about 12 nt to about 30 nt, from about 12 nt to about 25 nt, from about 12 nt to about 20 nt, from about 12 nt to about 19 nt, from about 19 nt to about 20 nt, from about 19 nt to about 25 nt, from about 19 nt to about 30 nt, from about 19 nt to about 35 nt, from about 19 nt to about 40 nt, from about 19 nt to about 45 nt, from about 19 nt to about 50 nt, from about 19 nt to
  • the DNA-targeting sequence of the DNA-targeting segment that is complementary to a target sequence of the target DNA is 20 nucleotides in length. In some cases, the DNA-targeting sequence of the DNA-targeting segment that is complementary to a target sequence of the target DNA is 16 nucleotides, 17 nucleotides, 18 nucleotides or 19 nucleotides in length.
  • the percent complementarity between the DNA-targeting sequence of the DNA-targeting segment and the target sequence of the target DNA can be at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%).
  • the DNA-targeting sequence may be at least about 80% identical to about 10 contiguous nucleotides, or at least about 80% identical to about 11 contiguous nucleotides, or at least about 80% identical to about 12 contiguous nucleotides, or at least about 80% identical to about 13 contiguous nucleotides, or at least about 80% identical to about 14 contiguous nucleotides, or at least about 80% identical to about 15 contiguous nucleotides, or at least about 80% identical to about 16 contiguous nucleotides, or at least about 80% identical to about 17 contiguous nucleotides of the target sequence.
  • the percent complementarity between the DNA-targeting sequence of the DNA-targeting segment and the target sequence of the target DNA is 100% over the seven contiguous 5′-most nucleotides of the target sequence of the complementary strand of the target DNA. In some cases, the percent complementarity between the DNA-targeting sequence of the DNA-targeting segment and the target sequence of the target DNA is at least 60% over about 20 contiguous nucleotides. In some cases, the percent complementarity between the DNA-targeting sequence of the DNA-targeting segment and the target sequence of the target DNA is 100% over the fourteen contiguous 5′-most nucleotides of the target sequence of the complementary strand of the target DNA and as low as 0% over the remainder.
  • the DNA-targeting sequence can be considered to be 14 nucleotides in length.
  • the percent complementarity between the DNA-targeting sequence of the DNA-targeting segment and the target sequence of the target DNA is 100% over the seven contiguous 5′-most nucleotides of the target sequence of the complementary strand of the target DNA and as low as 0% over the remainder.
  • the DNA-targeting sequence can be considered to be 7 nucleotides in length.
  • the protein-binding segment of a guide RNA interacts with a site-directed modifying polypeptide.
  • the guide RNA guides the bound polypeptide to a specific nucleotide sequence within target DNA via the above mentioned DNA-targeting segment.
  • the protein-binding segment of a guide RNA comprises two stretches of nucleotides that are complementary to one another. The complementary nucleotides of the protein-binding segment hybridize to form a double stranded RNA duplex (dsRNA).
  • a double-molecule guide RNA comprises two separate RNA molecules.
  • Each of the two RNA molecules of a double-molecule guide RNA comprises a stretch of nucleotides that are complementary to one another such that the complementary nucleotides of the two RNA molecules hybridize to form the double-stranded RNA duplex of the protein-binding segment.
  • the duplex-forming segment of the activator-RNA is at least about 60% identical to one of the activator-RNA (tracrRNA) molecules set forth in Supplementary Table S5, or a complement thereof, over a stretch of at least 8 contiguous nucleotides.
  • the duplex-forming segment of the activator-RNA (or the DNA encoding the duplex-forming segment of the activator-RNA) is at least about 60% identical, at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, or 100% identical, to one of the tracrRNA sequences set forth in Supplementary Table S5, or a complement thereof, over a stretch of at least 8 contiguous, at least 9 contiguous, at least 10 contiguous, at least 11 contiguous, at least 12 contiguous, at least 13 contiguous, at least 14 contiguous or at least 15 contiguous nucleotides.
  • the activator-RNA may comprise a nucleotide sequence that is at least 70% identical over at least 10 contiguous nucleotides, at least 80% identical over at least 10 contiguous nucleotides, at least 70% identical over at least 11 contiguous nucleotides, at least 80% identical over at least 11 contiguous nucleotides, at least 70% identical over at least 12 contiguous nucleotides, or at least 80% identical over at least 12 contiguous nucleotides of one of the tracrRNA sequences set forth in Supplementary Table S5.
  • the duplex-forming segment of the targeter-RNA is at least about 60% identical to one of the targeter-RNA (crRNA/CRISPR repeat) sequences set forth in Supplementary Table S5, or a complement thereof, over a stretch of at least 8 contiguous nucleotides.
  • the duplex-forming segment of the targeter-RNA (or the DNA encoding the duplex-forming segment of the targeter-RNA) is at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical or 100% identical to one of the crRNA/CRISPR repeat sequences set forth in Supplementary Table S5, or a complement thereof, over a stretch of at least 8 contiguous, at least 9 contiguous, at least 10 contiguous, at least 11 contiguous, at least 12 contiguous, at least 13 contiguous, at least 14 contiguous or at least 15 contiguous nucleotides.
  • the targeter-RNA may comprise a nucleotide sequence that is at least 70% identical over at least 10 contiguous nucleotides, at least 80% identical over at least 10 contiguous nucleotides, at least 70% identical over at least 11 contiguous nucleotides, at least 80% identical over at least 11 contiguous nucleotides, at least 70% identical over at least 12 contiguous nucleotides, at least 80% identical over at least 12 contiguous nucleotides, at least 80% identical over at least 13 contiguous nucleotides, at least 80% identical over at least 14 contiguous nucleotides, at least 80% identical over at least 15 contiguous nucleotides, at least 80% identical over at least 16 contiguous nucleotides, or at least 80% identical over at least 17 contiguous nucleotides, to one of the CRISPR repeat sequences set forth in Supplementary Table S5.
  • a two-molecule guide RNA can be designed to allow for controlled (i.e., conditional) binding of a targeter-RNA with an activator-RNA. Because a two-molecule guide RNA is not functional unless both the activator-RNA and the targeter-RNA are bound in a functional complex with Cas9, a two-molecule guide RNA can be inducible (e.g., drug inducible) by rendering the binding between the activator-RNA and the targeter-RNA to be inducible.
  • RNA aptamers can be used to regulate (i.e., control) the binding of the activator-RNA with the targeter-RNA. Accordingly, the activator-RNA and/or the targeter-RNA can comprise an RNA aptamer sequence.
  • RNA aptamers are known in the art and are generally a synthetic version of a riboswitch.
  • the terms “RNA aptamer” and “riboswitch” are used interchangeably herein to encompass both synthetic and natural nucleic acid sequences that provide for inducible regulation of the structure (and therefore the availability of specific sequences) of the RNA molecule of which they are part.
  • RNA aptamers usually comprise a sequence that folds into a particular structure (e.g., a hairpin), which specifically binds a particular drug (e.g., a small molecule). Binding of the drug causes a structural change in the folding of the RNA, which changes a feature of the nucleic acid of which the aptamer is a part.
  • an activator-RNA with an aptamer may not be able to bind to the cognate targeter-RNA unless the aptamer is bound by the appropriate drug;
  • a targeter-RNA with an aptamer may not be able to bind to the cognate activator-RNA unless the aptamer is bound by the appropriate drug;
  • a targeter-RNA and an activator-RNA, each comprising a different aptamer that binds a different drug may not be able to bind to each other unless both drugs are present.
  • a two-molecule guide RNA can be designed to be inducible.
  • aptamers and riboswitches can be found, for example, in: Nakamura et al., Genes Cells. 2012 May; 17(5):344-64; Vavalle et al., Future Cardiol. 2012 May; 8(3):371-82; Citartan et al., Biosens Bioelectron. 2012 April 15; 34(1):1-11; and Liberman et al., Wiley Interdiscip Rev RNA. 2012 May-June; 3(3):369-84; all of which are herein incorporated by reference in their entirety.
  • Non-limiting examples of nucleotide sequences that can be included in a two-molecule guide RNA include either of the sequences set forth in Supplementary Table S5, or complements thereof pairing with any sequences set forth in Supplementary Table S5, or complements thereof that can hybridize to form a protein binding segment.
  • a single-molecule guide RNA comprises two stretches of nucleotides (a targeter-RNA and an activator-RNA) that are complementary to one another, are covalently linked (directly, or by intervening nucleotides referred to as “linkers” or “linker nucleotides”), and hybridize to form the double stranded RNA duplex (dsRNA duplex) of the protein-binding segment, thus resulting in a stem-loop structure.
  • the targeter-RNA and the activator-RNA can be covalently linked via the 3′ end of the targeter-RNA and the 5′ end of the activator-RNA.
  • targeter-RNA and the activator-RNA can be covalently linked via the 5′ end of the targeter-RNA and the 3′ end of the activator-RNA.
  • the linker of a single-molecule guide RNA can have a length of from about 3 nucleotides to about 100 nucleotides.
  • the linker can have a length of from about 3 nucleotides (nt) to about 90 nt, from about 3 nucleotides (nt) to about 80 nt, from about 3 nucleotides (nt) to about 70 nt, from about 3 nucleotides (nt) to about 60 nt, from about 3 nucleotides (nt) to about 50 nt, from about 3 nucleotides (nt) to about 40 nt, from about 3 nucleotides (nt) to about 30 nt, from about 3 nucleotides (nt) to about 20 nt or from about 3 nucleotides (nt) to about 10 nt.
  • the linker can have a length of from about 3 nt to about 5 nt, from about 5 nt to about 10 nt, from about 10 nt to about 15 nt, from about 15 nt to about 20 nt, from about 20 nt to about 25 nt, from about 25 nt to about 30 nt, from about 30 nt to about 35 nt, from about 35 nt to about 40 nt, from about 40 nt to about 50 nt, from about 50 nt to about 60 nt, from about 60 nt to about 70 nt, from about 70 nt to about 80 nt, from about 80 nt to about 90 nt, or from about 90 nt to about 100 nt.
  • the linker of a single-molecule guide RNA is 4 nt.
  • An exemplary single-molecule guide RNA comprises two complementary stretches of nucleotides that hybridize to form a dsRNA duplex.
  • one of the two complementary stretches of nucleotides of the single-molecule guide RNA (or the DNA encoding the stretch) is at least about 60% identical to one of the activator-RNA (tracrRNA) molecules set forth in Supplementary Table S5, or a complement thereof, over a stretch of at least 8 contiguous nucleotides.
  • one of the two complementary stretches of nucleotides of the single-molecule guide RNA is at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical or 100° A) identical to one of the tracrRNA sequences set forth in Supplementary Table S5, or a complement thereof, over a stretch of at least 8 contiguous, at least 9 contiguous, at least 10 contiguous, at least 11 contiguous, at least 12 contiguous, at least 13 contiguous, at least 14 contiguous or at least 15 contiguous nucleotides.
  • the single-molecule guide RNA may comprise a nucleotide sequence that is at least 70% identical over at least 10 contiguous nucleotides, at least 80% identical over at least 10 contiguous nucleotides, at least 70% identical over at least 11 contiguous nucleotides, at least 80% identical over at least 11 contiguous nucleotides, at least 70% identical over at least 12 contiguous nucleotides, or at least 80% identical over at least 12 contiguous nucleotides of one of the tracrRNA sequences set forth in Supplementary Table S5.
  • one of the two complementary stretches of nucleotides of the single-molecule guide RNA is at least about 60% identical to one of the targeter-RNA (crRNA/CRISPR repeat) sequences set forth in Supplementary Table S5, or a complement thereof, over a stretch of at least 8 contiguous nucleotides.
  • one of the two complementary stretches of nucleotides of the single-molecule guide RNA is at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical or 100% identical to one of the crRNA/CRISPR repeat sequences set forth in Supplementary Table S5, or a complement thereof, over a stretch of at least 8 contiguous, at least 9 contiguous, at least 10 contiguous, at least 11 contiguous, at least 12 contiguous, at least 13 contiguous, at least 14 contiguous or at least 15 contiguous nucleotides.
  • the single-molecule guide RNA may comprise a nucleotide sequence that is at least 70% identical over at least 10 contiguous nucleotides, at least 80% identical over at least 10 contiguous nucleotides, at least 70% identical over at least 11 contiguous nucleotides, at least 80% identical over at least 11 contiguous nucleotides, at least 70% identical over at least 12 contiguous nucleotides, or at least 80% identical over at least 12 contiguous nucleotides, or at least about 80% identical to about 13 contiguous nucleotides, or at least about 80% identical to about 14 contiguous nucleotides, or at least about 80% identical to about 15 contiguous nucleotides, or at least about 80% identical to about 16 contiguous nucleotides, or at least about 80% identical to about 17 contiguous nucleotides of one of the CRISPR repeat sequences set forth in Supplementary Table S5.
  • each RNA is from a Cas9 cluster herein wherein the Cas9 endonucleases share 80% identity over 80% of their amino acid sequences.
  • RNA folding structure of a naturally occurring protein-binding domain of a DNA-targeting RNA can be taken into account in order to design artificial protein-binding domains (either two-molecule or single-molecule versions).
  • an artificial DNA-targeting-RNA can be designed to mimic the natural structure for a given species when using the Cas9 (or a related Cas9) from that species.
  • a suitable guide RNA can be an artificially designed RNA (non-naturally occurring) comprising a protein-binding domain that was designed to mimic the structure of a protein-binding domain of a naturally occurring guide RNA.
  • the protein-binding segment can have a length of from about 10 nucleotides to about 100 nucleotides.
  • the protein-binding segment can have a length of from about 15 nucleotides (nt) to about 80 nt, from about 15 nt to about 50 nt, from about 15 nt to about 40 nt, from about 15 nt to about 30 nt or from about 15 nt to about 25 nt.
  • the dsRNA duplex of the protein-binding segment can have a length from about 6 base pairs (bp) to about 50 bp.
  • the dsRNA duplex of the protein-binding segment can have a length from about 6 bp to about 40 bp, from about 6 bp to about 30 bp, from about 6 bp to about 25 bp, from about 6 bp to about 20 bp, from about 6 bp to about 15 bp, from about 8 bp to about 40 bp, from about 8 bp to about 30 bp, from about 8 bp to about 25 bp, from about 8 bp to about 20 bp or from about 8 bp to about 15 bp.
  • the dsRNA duplex of the protein-binding segment can have a length from about from about 8 bp to about 10 bp, from about 10 bp to about 15 bp, from about 15 bp to about 18 bp, from about 18 bp to about 20 bp, from about 20 bp to about 25 bp, from about 25 bp to about 30 bp, from about 30 bp to about 35 bp, from about 35 bp to about 40 bp, or from about 40 bp to about 50 bp.
  • the dsRNA duplex of the protein-binding segment has a length of 36 base pairs.
  • the percent complementarity between the nucleotide sequences that hybridize to form the dsRNA duplex of the protein-binding segment can be at least about 60%.
  • the percent complementarity between the nucleotide sequences that hybridize to form the dsRNA duplex of the protein-binding segment can be at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%.
  • the percent complementarity between the nucleotide sequences that hybridize to form the dsRNA duplex of the protein-binding segment is 100%.
  • a guide RNA and a site-directed modifying polypeptide form a complex.
  • the guide RNA provides target specificity to the complex by comprising a nucleotide sequence that is complementary to a sequence of a target DNA (as noted above).
  • the site-directed modifying polypeptide is guided to a DNA sequence (e.g. a chromosomal sequence or an extrachromosomal sequence, e.g. an episomal sequence, a minicircle sequence, a mitochondrial sequence, a chloroplast sequence, etc.) by virtue of its association with at least the protein-binding segment of the guide RNA (described above).
  • a site-directed modifying polypeptide modifies target DNA (e.g., cleavage or methylation of target DNA) and/or a polypeptide associated with target DNA (e.g., methylation or acetylation of a histone tail).
  • a site-directed modifying polypeptide is also referred to herein as a “site-directed polypeptide” or an “RNA binding site-directed modifying polypeptide.” In some cases, the site-directed modifying polypeptide is a naturally-occurring modifying polypeptide.
  • the site-directed modifying polypeptide is not a naturally-occurring polypeptide (e.g., a chimeric polypeptide as discussed below or a naturally-occurring polypeptide that is modified, e.g., mutation, deletion, insertion).
  • Naturally-occurring site-directed modifying polypeptides bind a guide RNA, are thereby directed to a specific sequence within a target DNA, and cleave the target DNA to generate a double strand break.
  • the amino acid sequences of exemplary naturally-occurring Cas9 site-directed modifying polypeptide orthologs are set out in SEQ ID NOs: 1-800.
  • the amino acid sequence of the S. pyrogens Cas9 endonuclease is set out in SEQ ID NO: 8.
  • a site-directed modifying polypeptide comprises two portions, an RNA-binding portion and an activity portion.
  • a site-directed modifying polypeptide comprises: (i) an RNA-binding portion that interacts with a guide RNA, wherein the guide RNA comprises a nucleotide sequence that is complementary to a sequence in a target DNA; and (ii) an activity portion that exhibits site-directed enzymatic activity (e.g., activity for DNA methylation, activity for DNA cleavage, activity for histone acetylation, activity for histone methylation, etc.), wherein the site of enzymatic activity is determined by the guide RNA.
  • site-directed enzymatic activity e.g., activity for DNA methylation, activity for DNA cleavage, activity for histone acetylation, activity for histone methylation, etc.
  • a site-directed modifying polypeptide comprises: (i) an RNA-binding portion that interacts with a guide RNA, wherein the guide RNA comprises a nucleotide sequence that is complementary to a sequence in a target DNA; and (ii) an activity portion that modulates transcription within the target DNA (e.g., to increase or decrease transcription), wherein the site of modulated transcription within the target DNA is determined by the guide RNA.
  • a site-directed modifying polypeptide has enzymatic activity that modifies target DNA (e.g., nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylase activity).
  • target DNA e.g., nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity
  • a site-directed modifying polypeptide has enzymatic activity that modifies a polypeptide (e.g., a histone) associated with target DNA (e.g., methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity or demyristoylation activity).
  • a polypeptide e.g., a histone
  • target DNA e.g., methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity
  • the site-directed modifying polypeptide comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%, amino acid sequence identity to amino acids 7-166 and/or 731-1003 of SEQ ID NO: 8, or to the corresponding portions in any of the amino acid sequences set forth as SEQ ID NOs: 1-800.
  • a nucleic acid (e.g., a guide RNA) comprises one or more modifications, e.g., a base modification, a backbone modification, etc, to provide the nucleic acid with a new or enhanced feature (e.g., improved stability).
  • a nucleoside is a base-sugar combination.
  • the base portion of the nucleoside is normally a heterocyclic base.
  • the two most common classes of such heterocyclic bases are the purines and the pyrimidines.
  • Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside.
  • the phosphate group can be linked to the 2′, the 3′, or the 5′ hydroxyl moiety of the sugar.
  • the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound.
  • the respective ends of this linear polymeric compound can be further joined to form a circular compound, however, linear compounds are generally suitable.
  • linear compounds may have internal nucleotide base complementarity and may therefore fold in a manner as to produce a fully or partially double-stranded compound.
  • the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide.
  • the normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.
  • nucleic acids containing modifications include nucleic acids containing modified backbones or non-natural internucleoside linkages.
  • Nucleic acids having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.
  • Suitable modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 1 -amino phosphoramidate and aminoalkylphosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5
  • Suitable oligonucleotides having inverted polarity comprise a single 3′ to 3′ linkage at the 3-most internucleotide linkage i.e. a single inverted nucleoside residue which may be a basic (the nucleobase is missing or has a hydroxyl group in place thereof).
  • Various salts such as, for example, potassium or sodium), mixed salts and free acid forms are also included.
  • a nucleic acid comprises one or more phosphorothioate and/or heteroatom internucleoside linkages, in particular —CH 2 —NH—O—CH 2 —, —CH 2 —N(CH 3 )—O—CH 2 — (known as a methylene (methylimino) or MMI backbone), —CH 2 —O—N(CH 3 )—CH 2 —, —CH 2 —N(CH 3 )—N(CH 3 )—CH 2 — and —O—N(CH 3 )—CH 2 —CH 2 — (wherein the native phosphodiester internucleotide linkage is represented as —O—P( ⁇ O)(OH)—O—CH 2 —).
  • MMI type internucleoside linkages are disclosed in the above referenced U.S. Pat. No. 5,489,677. Suitable amide internucleoside linkages are disclosed in t U.S. Pat. No. 5,602,240
  • nucleic acids having morpholino backbone structures as described in, e.g., U.S. Pat. No. 5,034,506.
  • a nucleic acid comprises a 6-membered morpholino ring in place of a ribose ring.
  • a phosphorodiamidate or other non-phosphodiester internucleoside linkage replaces a phosphodiester linkage.
  • Suitable modified polynucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • morpholino linkages formed in part from the sugar portion of a nucleoside
  • siloxane backbones sulfide, sulfoxide and sulfone backbones
  • formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
  • riboacetyl backbones alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH 2 component parts.
  • a nucleic acid can be a nucleic acid mimetic.
  • the term “mimetic” as it is applied to polynucleotides is intended to include polynucleotides wherein only the furanose ring or both the furanose ring and the internucleotide linkage are replaced with non-furanose groups, replacement of only the furanose ring is also referred to in the art as being a sugar surrogate.
  • the heterocyclic base moiety or a modified heterocyclic base moiety is maintained for hybridization with an appropriate target nucleic acid.
  • One such nucleic acid, a polynucleotide mimetic that has been shown to have excellent hybridization properties is referred to as a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • the sugar-backbone of a polynucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone.
  • the nucleotides are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • PNA peptide nucleic acid
  • the backbone in PNA compounds is two or more linked aminoethylglycine units which gives PNA an amide containing backbone.
  • the heterocyclic base moieties are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • Representative U.S. patents that describe the preparation of PNA compounds include, but are not limited to: U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262.
  • Another class of polynucleotide mimetic that has been studied is based on linked morpholino units (morpholino nucleic acid) having heterocyclic bases attached to the morpholino ring.
  • a number of linking groups have been reported that link the morpholino monomeric units in a morpholino nucleic acid.
  • One class of linking groups has been selected to give a non-ionic oligomeric compound.
  • the non-ionic morpholino-based oligomeric compounds are less likely to have undesired interactions with cellular proteins.
  • Morpholino-based polynucleotides are nonionic mimics of oligonucleotides which are less likely to form undesired interactions with cellular proteins (Dwaine A. Braasch and David R.
  • Morpholino-based polynucleotides are disclosed in U.S. Pat. No. 5,034,506. A variety of compounds within the morpholino class of polynucleotides have been prepared, having a variety of different linking groups joining the monomeric subunits.
  • CeNA cyclohexenyl nucleic acids
  • the furanose ring normally present in a DNA/RNA molecule is replaced with a cyclohexenyl ring.
  • CeNA DMT protected phosphoramidite monomers have been prepared and used for oligomeric compound synthesis following classical phosphoramidite chemistry.
  • Fully modified CeNA oligomeric compounds and oligonucleotides having specific positions modified with CeNA have been prepared and studied (see Wang et al., J. Am. Chem. Soc., 2000, 122, 85958602).
  • the incorporation of CeNA monomers into a DNA chain increases its stability of a DNA/RNA hybrid.
  • CeNA oligoadenylates formed complexes with RNA and DNA complements with similar stability to the native complexes.
  • the study of incorporating CeNA structures into natural nucleic acid structures was shown by NMR and circular dichroism to proceed with easy conformational adaptation.
  • a further modification includes Locked Nucleic Acids (LNAs) in which the 2′-hydroxyl group is linked to the 4′ carbon atom of the sugar ring thereby forming a 2′-C,4′-C-oxymethylene linkage thereby forming a bicyclic sugar moiety.
  • the linkage can be a methylene (—CH 2 —), group bridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2 (Singh et al., Chem. Commun., 1998, 4, 455-456).
  • Potent and nontoxic antisense oligonucleotides containing LNAs have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638).
  • LNA monomers adenine, cytosine, guanine, 5-methylcytosine, thymine and uracil, along with their oligomerization, and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). LNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226.
  • a nucleic acid can also include one or more substituted sugar moieties.
  • Suitable polynucleotides comprise a sugar substituent group selected from: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C.sub.1 to C 10 alkyl or C 2 to C 10 alkenyl and alkynyl.
  • Suitable polynucleotides comprise a sugar substituent group selected from: C 1 to C 10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties.
  • a sugar substituent group selected from: C 1 to C 10 lower alkyl,
  • a suitable modification includes 2′-methoxyethoxy 2′-O—CH 2 CH 2 OCH 3 , also known as -2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Hely. Chinn. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group.
  • a further suitable modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH 2 ) 2 ON(CH 3 ) 2 group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O—CH 2 —O—CH 2 —N(CH 3 ) 2 .
  • sugar substituent groups include methoxy (—O—CH 3 ), aminopropoxy (—O—CH 2 CH 2 CH 2 NH 2 ), allyl (—CH 2 —CH ⁇ CH 2 ), —O-allyl(-O—CH 2 —CH ⁇ CH 2 ) and fluoro (F).
  • 2′-sugar substituent groups may be in the arabino (up) position or ribo (down) position.
  • a suitable 2′-arabino modification is 2′-F.
  • Similar modifications may also be made at other positions on the oligomeric compound, particularly the 3′ position of the sugar on the 3′ terminal nucleoside or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide.
  • Oligomeric compounds may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
  • a nucleic acid may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C ⁇ C—CH 3 ) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and gu
  • nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g.
  • Heterocyclic base moieties may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.
  • Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.
  • nucleobases are useful for increasing the binding affinity of an oligomeric compound.
  • These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C.
  • “Complementary” refers to the capacity for pairing, through base stacking and specific hydrogen bonding, between two sequences comprising naturally or non-naturally occurring (e.g., modified as described above) bases (nucleosides) or analogs thereof. For example, if a base at one position of a nucleic acid is capable of hydrogen bonding with a base at the corresponding position of a target, then the bases are considered to be complementary to each other at that position. Nucleic acids can comprise universal bases, or inert abasic spacers that provide no positive or negative contribution to hydrogen bonding. Base pairings may include both canonical Watson-Crick base pairing and non-Watson-Crick base pairing (e.g., Wobble base pairing and Hoogsteen base pairing).
  • adenosine-type bases are complementary to thymidine-type bases (T) or uracil-type bases (U), that cytosine-type bases (C) are complementary to guanosine-type bases (G), and that universal bases such as such as 3-nitropyrrole or 5-nitroindole can hybridize to and are considered complementary to any A, C, U, or T.
  • T thymidine-type bases
  • U uracil-type bases
  • C cytosine-type bases
  • G guanosine-type bases
  • universal bases such as such as 3-nitropyrrole or 5-nitroindole
  • nucleic acid Another possible modification of a nucleic acid involves chemically linking to the polynucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide.
  • moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups.
  • Conjugate groups include, but are not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers.
  • Suitable conjugate groups include, but are not limited to, cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.
  • Groups that enhance the pharmacodynamic properties include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid.
  • Groups that enhance the pharmacokinetic properties include groups that improve uptake, distribution, metabolism or excretion of a nucleic acid.
  • Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem.
  • lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053
  • a conjugate may include a “Protein Transduction Domain” or PTD (also known as a CPP—cell penetrating peptide), which may refer to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates traversing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane.
  • PTD Protein Transduction Domain
  • a PTD attached to another molecule which can range from a small polar molecule to a large macromolecule and/or a nanoparticle, facilitates the molecule traversing a membrane, for example going from extracellular space to intracellular space, or cytosol to within an organelle.
  • a PTD is covalently linked to the amino terminus of an exogenous polypeptide (e.g., a site-directed modifying polypeptide). In some embodiments, a PTD is covalently linked to the carboxyl terminus of an exogenous polypeptide (e.g., a site-directed modifying polypeptide). In some embodiments, a PTD is covalently linked to a nucleic acid (e.g., a guide RNA, a polynucleotide encoding a guide RNA, a polynucleotide encoding a site-directed modifying polypeptide, etc.).
  • a nucleic acid e.g., a guide RNA, a polynucleotide encoding a guide RNA, a polynucleotide encoding a site-directed modifying polypeptide, etc.
  • Exemplary PTDs include but are not limited to a minimal undecapeptide protein transduction domain (corresponding to residues 47-57 of HIV-1 TAT comprising YGRKKRRQRRR; a polyarginine sequence comprising a number of arginines sufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); an Drosophila Antennapedia protein transduction domain (Noguchi et al. (2003) Diabetes 52(7):1732-1737); a truncated human calcitonin peptide (Trehin et al. (2004) Pharm.
  • a minimal undecapeptide protein transduction domain corresponding to residues 47-57 of HIV-1 TAT comprising YGRKKRRQRRR
  • a polyarginine sequence comprising a number of arginines sufficient to direct entry into a cell (e.g.,
  • Exemplary PTDs include but are not limited to, YGRKKRRQRRR; RKKRRQRRR; an arginine homopolymer of from 3 arginine residues to 50 arginine residues;
  • Exemplary PTD domain amino acid sequences include, but are not limited to, any of the following: YGRKKRRQRRR; RKKRRQRR; YARAAARQARA; THRLPRRRRRR; and GGRRARRRRRR.
  • the PTD is an activatable CPP (ACPP) (Aguilera et al. (2009) Integr Biol ( Camb ) June; 1(5-6): 371-381).
  • ACPPs comprise a polycationic CPP (e.g., Arg9 or “R9”) connected via a cleavable linker to a matching polyanion (e.g., Glu9 or “E9”), which reduces the net charge to nearly zero and thereby inhibits adhesion and uptake into cells.
  • a polycationic CPP e.g., Arg9 or “R9”
  • a matching polyanion e.g., Glu9 or “E9”
  • a guide RNA comprises two separate RNA polynucleotide molecules.
  • the first of the two separate RNA polynucleotide molecules comprises a nucleotide sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% nucleotide sequence identity over a stretch of at least 8 contiguous, at least 9 contiguous, at least 10 contiguous, at least 11 contiguous, at least 12 contiguous, at least 13 contiguous, at least 14 contiguous or at least 15 contiguous nucleotides to any one of the tracrRNA nucleotide sequences set forth in Supplementary Table S5, or complements thereof.
  • the second of the two separate RNA polynucleotide molecules comprises a nucleotide sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% nucleotide sequence identity over a stretch of at least 8 contiguous, at least 9 contiguous, at least 10 contiguous, at least 11 contiguous, at least 12 contiguous, at least 13 contiguous, at least 14 contiguous or at least 15 contiguous nucleotides to the cognate CRISPR repeat nucleotide sequence set forth in Supplementary Table S5, or complements thereof.
  • a suitable guide RNA is a single-molecule RNA polynucleotide and comprises a first nucleotide sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% nucleotide sequence identity over a stretch of at least 8 contiguous, at least 9 contiguous, at least 10 contiguous, at least 11 contiguous, at least 12 contiguous, at least 13 contiguous, at least 14 contiguous or at least 15 contiguous nucleotides to any one of the tracrRNA nucleotide sequences set forth in Supplementary Table S5 and a second nucleotide sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%,
  • the single-molecule guide RNAs comprise a DNA-targeting segment and a protein-binding segment complementary thereto, wherein the protein-binding segment comprises a tracrRNA set out in Supplementary Table S5 or wherein the protein-binding segment comprises a tracrRNA at least 80% identical over at least 20 nucleotides to a tracrRNA set out in Supplementary Table S5, or at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% nucleotide sequence identity over a stretch of at least 8 contiguous, at least 9 contiguous, at least 10 contiguous, at least 11 contiguous, at least 12 contiguous, at least 13 contiguous, at least 14 contiguous or at least 15 contiguous nucleotides of any one of the tracrRNA nucleotide sequences set forth in Supplementary Table
  • the protein-binding segment may comprise a tracrRNA at least 70% identical over at least 10 contiguous nucleotides, at least 80% identical over at least 10 contiguous nucleotides, at least 70% identical over at least 11 contiguous nucleotides, at least 80% identical over at least 11 contiguous nucleotides, at least 70% identical over at least 12 contiguous nucleotides, or at least 80% identical over at least 12 contiguous nucleotides.
  • the single-molecule guide RNAs comprise a DNA-targeting segment and a protein-binding segment, wherein the protein-binding segment comprises a tracrRNA set out in Supplementary Table S5 or wherein the protein-binding segment comprises a tracrRNA at least 80% identical over at least 20 nucleotides to a tracrRNA set out in Supplementary Table S5.
  • the protein-binding segment comprises a CRISPR repeat set out in Supplementary Table S5 that is the CRISPR repeat cognate to the tracrRNA of the protein-binding segment.
  • the DNA-targeting segment comprises RNA complementary to a protospacer-like sequence in a target DNA 5′ to a PAM sequence.
  • the tracrRNA and CRISPR repeat are respectively the C. jejuni tracrRNA and its cognate CRISPR repeat set out in Supplementary Table S5 and the PAM sequence is NNNNACA. In some embodiments, the tracrRNA and CRISPR repeat are respectively at least 80% identical to the C. jejuni tracrRNA and its cognate CRISPR repeat set out in Supplementary Table S5 and the PAM sequence is NNNNACA. In some embodiments, the single-molecule guide RNA comprises a sequence that hybridizes to a protospacer-like sequence set out in one of SEQ ID NOs: 801-2701.
  • the double-molecule guide RNAs comprise a targeter-RNA and an activator-RNA complementary thereto, wherein the activator-RNA comprises a tracrRNA set out in Supplementary Table S5 or wherein the activator-RNA comprises a tracrRNA at least 80% identical over at least 20 nucleotides to a tracrRNA set out in Supplementary Table S5.
  • the double-molecule guide RNA comprises a modified backbone, a non-natural internucleoside linkage, a nucleic acid mimetic, a modified sugar moiety, a base modification, a modification or sequence that provides for modified or regulated stability, a modification or sequence that provides for subcellular tracking, a modification or sequence that provides for tracking, or a modification or sequence that provides for a binding site for a protein or protein complex.
  • the targeter-RNA comprises a CRISPR repeat set out in Supplementary Table S5.
  • the targeter-RNA comprises a CRISPR repeat set out in Supplementary Table S5 that is the cognate CRISPR repeat of the tracrRNA of the activator-RNA.
  • the targeter-RNA further comprises RNA complementary to a protospacer-like sequence in a target DNA 5′ to a PAM sequence.
  • the tracrRNA and CRISPR repeat are respectively the C. jejuni tracrRNA and its cognate CRISPR repeat set out in Supplementary Table S5 and the PAM sequence is NNNNACA.
  • the tracrRNA and CRISPR repeat are at least 80% identical to respectively the C. jejuni tracrRNA and its cognate CRISPR repeat set out in Supplementary Table S5 and the PAM sequence is NNNNACA.
  • the double-molecule guide RNA comprises a sequence that hybridizes to a protospacer-like sequence set out in one of SEQ ID NOs: 801-2701.
  • a nucleic acid comprising a nucleotide sequence encoding a guide RNA and/or a site-directed modifying polypeptide.
  • a guide RNA-encoding nucleic acid is an expression vector, e.g., a recombinant expression vector.
  • a method involves contacting a target DNA or introducing into a cell (or a population of cells) one or more nucleic acids comprising nucleotide sequences encoding a guide RNA and/or a site-directed modifying polypeptide.
  • a cell comprising a target DNA is in vitro.
  • a cell comprising a target DNA is in vivo.
  • Suitable nucleic acids comprising nucleotide sequences encoding a guide RNA and/or a site-directed modifying polypeptide include expression vectors, where an expression vector comprising a nucleotide sequence encoding a guide RNA and/or a site-directed modifying polypeptide is a “recombinant expression vector.”
  • the recombinant expression vector is a viral construct, e.g., a recombinant adeno-associated virus construct (see, e.g., U.S. Pat. No. 7,078,387), a recombinant adenoviral construct, a recombinant lentiviral construct, a recombinant retroviral construct, etc.
  • a viral construct e.g., a recombinant adeno-associated virus construct (see, e.g., U.S. Pat. No. 7,078,387), a recombinant adenoviral construct, a recombinant lentiviral construct, a recombinant retroviral construct, etc.
  • Suitable expression vectors include, but are not limited to, viral vectors (e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis
  • SV40 herpes simplex virus
  • human immunodeficiency virus see, e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816, 1999
  • a retroviral vector e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus
  • retroviral vector e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myelop
  • Suitable expression vectors are known to those of skill in the art, and many are commercially available.
  • the following vectors are provided by way of example; for eukaryotic host cells: pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia).
  • any other vector may be used so long as it is compatible with the host cell.
  • any of a number of suitable transcription and translation control elements including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector (see e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544).
  • a nucleotide sequence encoding a guide RNA and/or a site-directed modifying polypeptide is operably linked to a control element, e.g., a transcriptional control element, such as a promoter.
  • a control element e.g., a transcriptional control element, such as a promoter.
  • the transcriptional control element may be functional in either a eukaryotic cell, e.g., a mammalian cell; or a prokaryotic cell (e.g., bacterial or archaeal cell).
  • a nucleotide sequence encoding a guide RNA and/or a site-directed modifying polypeptide is operably linked to multiple control elements that allow expression of the nucleotide sequence encoding a guide RNA and/or a site-directed modifying polypeptide in both prokaryotic and eukaryotic cells.
  • eukaryotic promoters include those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retrovirus, and mouse metallothionein-l. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
  • the expression vector may also contain a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector may also include appropriate sequences for amplifying expression.
  • the expression vector may also include nucleotide sequences encoding protein tags (e.g., 6 ⁇ His tag, hemagglutinin tag, green fluorescent protein, etc.) that are fused to the site-directed modifying polypeptide, thus resulting in a chimeric polypeptide.
  • protein tags e.g., 6 ⁇ His tag, hemagglutinin tag, green fluorescent protein, etc.
  • a nucleotide sequence encoding a guide RNA and/or a site-directed modifying polypeptide is operably linked to an inducible promoter. In some embodiments, a nucleotide sequence encoding a guide RNA and/or a site-directed modifying polypeptide is operably linked to a constitutive promoter.
  • nucleic acid e.g., an expression construct
  • Suitable methods include e.g., viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, nanoparticle-mediated nucleic acid delivery (see, e.g., Panyam et., al Adv Drug Deliv Rev. 2012 Sep. 13. pii: S0169-409X(12)00283-9. doi: 10.1016/j.addr.2012.09.023), and the like.
  • PKI polyethyleneimine
  • the present disclosure provides a chimeric site-directed modifying polypeptide.
  • a chimeric site-directed modifying polypeptide interacts with (e.g., binds to) a guide RNA (described above).
  • the guide RNA guides the chimeric site-directed modifying polypeptide to a target sequence within target DNA (e.g. a chromosomal sequence or an extrachromosomal sequence, e.g. an episomal sequence, a minicircle sequence, a mitochondrial sequence, a chloroplast sequence, etc.).
  • a chimeric site-directed modifying polypeptide modifies target DNA (e.g., cleavage or methylation of target DNA) and/or a polypeptide associated with target DNA (e.g., methylation or acetylation of a histone tail).
  • a chimeric site-directed modifying polypeptide modifies target DNA (e.g., cleavage or methylation of target DNA) and/or a polypeptide associated with target DNA (e.g., methylation or acetylation of a histone tail).
  • a chimeric site-directed modifying polypeptide is also referred to herein as a “chimeric site-directed polypeptide” or a “chimeric RNA binding site-directed modifying polypeptide.”
  • a chimeric site-directed modifying polypeptide comprises two portions, an RNA-binding portion and an activity portion.
  • a chimeric site-directed modifying polypeptide comprises amino acid sequences that are derived from at least two different polypeptides.
  • a chimeric site-directed modifying polypeptide can comprise modified and/or naturally-occurring polypeptide sequences (e.g., a first amino acid sequence from a modified or unmodified Cas9 protein; and a second amino acid sequence other than the Cas9 protein).
  • the RNA-binding portion of a chimeric site-directed modifying polypeptide is a naturally-occurring polypeptide. In other cases, the RNA-binding portion of a chimeric site-directed modifying polypeptide is not a naturally-occurring molecule (modified, e.g., mutation, deletion, insertion).
  • Naturally-occurring RNA-binding portions of interest are derived from site-directed modifying polypeptides known in the art. For example, SEQ ID NOs: 1-800 provide a non-limiting set of naturally occurring Cas9 endonucleases that can be used as site-directed modifying polypeptides.
  • the RNA-binding portion of a chimeric site-directed modifying polypeptide comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to the RNA-binding portion of a polypeptide set forth in SEQ ID NOs: 1-800.
  • the site-directed modifying polypeptide comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%, amino acid sequence identity to amino acids 7-166 and/or 731-1003 of SEQ ID NO: 8, or to the corresponding portions in any of the amino acid sequences set forth as SEQ ID NOs: 1-800.
  • the chimeric site-directed modifying polypeptide comprises an “activity portion.”
  • the activity portion of a chimeric site-directed modifying polypeptide comprises the naturally-occurring activity portion of a site-directed modifying polypeptide (e.g., Cas9 endonuclease).
  • the activity portion of a subject chimeric site-directed modifying polypeptide comprises a modified amino acid sequence (e.g., substitution, deletion, insertion) of a naturally-occurring activity portion of a site-directed modifying polypeptide.
  • Naturally-occurring activity portions of interest are derived from site-directed modifying polypeptides known in the art.
  • SEQ ID NOs: 1-800 are a non-limiting set of naturally occurring Cas9 endonucleases that can be used as site-directed modifying polypeptides.
  • the activity portion of a chimeric site-directed modifying polypeptide is variable and may comprise any heterologous polypeptide sequence that may be useful in the methods disclosed herein.
  • the activity portion of a site-directed modifying polypeptide comprises a portion of a Cas9 ortholog (including, but not limited to, the Cas9 orthologs set out in one of SEQ ID NOs: 1-800) that is at least 90% identical to amino acids 7-166 of SEQ ID NO: 8 and/or at least 90% identical to amino acids 731-1003 of SEQ ID NO: 8.
  • a chimeric site-directed modifying polypeptide comprises: (i) an RNA-binding portion that interacts with a guide RNA, wherein the guide RNA comprises a nucleotide sequence that is complementary to a sequence in a target DNA; and (ii) an activity portion that exhibits site-directed enzymatic activity (e.g., activity for DNA methylation, activity for DNA cleavage, activity for histone acetylation, activity for histone methylation, etc.), wherein the site of enzymatic activity is determined by the guide RNA.
  • site-directed enzymatic activity e.g., activity for DNA methylation, activity for DNA cleavage, activity for histone acetylation, activity for histone methylation, etc.
  • a chimeric site-directed modifying polypeptide comprises: (i) an RNA-binding portion that interacts with a guide RNA, wherein the guide RNA comprises a nucleotide sequence that is complementary to a sequence in a target DNA; and (ii) an activity portion that modulates transcription within the target DNA (e.g., to increase or decrease transcription), wherein the site of modulated transcription within the target DNA is determined by the guide RNA.
  • the activity portion of a chimeric site-directed modifying polypeptide has enzymatic activity that modifies target DNA (e.g., nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylase activity).
  • target DNA e.g., nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinas
  • the activity portion of a chimeric site-directed modifying polypeptide has enzymatic activity (e.g., methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity or demyristoylation activity) that modifies a polypeptide associated with target DNA (e.g., a histone).
  • enzymatic activity e.g., methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity
  • the activity portion of a chimeric site-directed modifying polypeptide exhibits enzymatic activity (described above). In other cases, the activity portion of a chimeric site-directed modifying polypeptide modulates transcription of the target DNA (described above).
  • the activity portion of a chimeric site-directed modifying polypeptide is variable and may comprise any heterologous polypeptide sequence that may be useful in the methods disclosed herein.
  • the activity portion of the chimeric site-directed modifying polypeptide comprises a modified form of the Cas9 protein, including modified forms of any of the Cas9 orthologs described herein, such as SEQ ID NOs: 1-800).
  • the modified form of the Cas9 protein comprises an amino acid change (e.g., deletion, insertion, or substitution) that reduces the naturally-occurring nuclease activity of the Cas9 protein.
  • the modified form of the Cas9 protein has less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nuclease activity of the corresponding wild-type Cas9 polypeptide.
  • the modified form of the Cas9 polypeptide has no substantial nuclease activity.
  • the modified form of the Cas9 polypeptide is a D10A (aspartate to alanine at amino acid position 10 of SEQ ID NO:8) mutation (or the corresponding mutation of any of the proteins presented in SEQ ID NOs: 1-800) that can cleave the complementary strand of the target DNA but has reduced ability to cleave the non-complementary strand of the target DNA.
  • the modified form of the SEQ ID NO: 8 Cas9 polypeptide is a H840A (histidine to alanine at amino acid position 840) mutation (or the corresponding mutation of any of the proteins set forth as SEQ ID NOs: 1-800) that can cleave the non-complementary strand of the target DNA but has reduced ability to cleave the complementary strand of the target DNA.
  • the modified form of the SEQ ID NO: 8 Cas9 polypeptide harbors both the D10A and the H840A mutations (or the corresponding mutations of any of the proteins set forth as SEQ ID NOs: 1-800) such that the polypeptide has a reduced ability to cleave both the complementary and the non-complementary strands of the target DNA.
  • Other residues can be mutated to achieve the above effects (i.e. inactivate one or the other nuclease portions).
  • pyogenes Cas9 residues D10, G12, G17, E762, H840, N863, H982, H983, A984, D986, and/or A987 of SEQ ID NO: 8 can be altered (i.e., substituted). Also, mutations other than alanine substitutions are contemplated.
  • a modified Cas9 endonuclease comprises one or more mutations corresponding to S. pyogenes Cas9 mutation E762A, HH983AA or D986A in SEQ ID NO: 8.
  • the modified Cas 9 endonuclease further comprises one or more mutations corresponding to S. pyogenes Cas9 mutation D10A, H840A, G12A, G17A, N854A, N863A, N982A or A984A in SEQ ID NO: 8.
  • the modified Cas9 endonuclease may comprise a variant at least about 75% identical to any of SEQ ID NOs: 1-800 that comprises one or more mutations corresponding to a mutation E762A, HH983AA or D986A in SEQ ID NO: 8; and/or one or more mutations corresponding to a mutation D10A, H840A, G12A, G17A, N854A, N863A, N982A or A984A in SEQ ID NO: 8.
  • such a variant comprises a region at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100% amino acid sequence identity to the regions corresponding to amino acids 7-166 and/or 731-1003 of SEQ ID NO: 8.
  • Table 1 lists four motifs that are present in Cas9 sequences from various species. The amino acids listed here are from the Cas9 from S. pyogenes (SEQ ID NO: 8). Motif Amino acids (residue #s) Highly conserved RuvC-like I IGLDIGTNSVGWAVI (7-21) D10, G12, G17 RuvC-like II IVIEMARE (759-766) E762 HNH-motif DVDHIVPQSFLKDDSIDNKVLTRSDKN (837- 863) H840, N854, N863 RuvC-like II HHAHDAYL (982-989) H982, H983, A984, D986, A987
  • the chimeric site-directed modifying polypeptide comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100% amino acid sequence identity to amino acids 7-166 and/or 731-1003 of SEQ ID NO: 8, or to the corresponding portions in any of the amino acid sequences set forth as SEQ ID NOs: 1-800.
  • the chimeric site-directed modifying polypeptide comprises 4 motifs (as listed in Table 1), each with amino acid sequences having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100% amino acid sequence identity to each of the 4 motifs listed in Table 1, or to the corresponding portions in any of the amino acid sequences set forth as SEQ ID NOs: 1-800.
  • the chimeric site-directed modifying polypeptide comprises amino acid sequences having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100% amino acid sequence identity to amino acids 7-166 and/or 731-1003 of SEQ ID NO: 8, or to the corresponding portions in any of the amino acid sequences set forth as SEQ ID NOs: 1-800.
  • the activity portion of the site-directed modifying polypeptide comprises a heterologous polypeptide that has DNA-modifying activity and/or transcription factor activity and/or DNA-associated polypeptide-modifying activity.
  • a heterologous polypeptide replaces a portion of the Cas9 polypeptide that provides nuclease activity.
  • a site-directed modifying polypeptide comprises both a portion of the Cas9 polypeptide that normally provides nuclease activity (and that portion can be fully active or can instead be modified to have less than 100% of the corresponding wild-type activity) and a heterologous polypeptide.
  • a chimeric site-directed modifying polypeptide is a fusion polypeptide comprising both the portion of the Cas9 polypeptide that normally provides nuclease activity and the heterologous polypeptide.
  • a chimeric site-directed modifying polypeptide is a fusion polypeptide comprising a modified variant of the activity portion of the Cas9 polypeptide (e.g., amino acid change, deletion, insertion) and a heterologous polypeptide.
  • a chimeric site-directed modifying polypeptide is a fusion polypeptide comprising a heterologous polypeptide and the RNA-binding portion of a naturally-occurring or a modified site-directed modifying polypeptide.
  • a naturally-occurring (or modified, e.g., mutation, deletion, insertion) bacterial Cas9 polypeptide may be fused to a heterologous polypeptide sequence (i.e. a polypeptide sequence from a protein other than Cas9 or a polypeptide sequence from another organism).
  • the heterologous polypeptide sequence may exhibit an activity (e.g., enzymatic activity) that will also be exhibited by the chimeric Cas9 protein (e.g., methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitinating activity, etc.).
  • a heterologous nucleic acid sequence may be linked to another nucleic acid sequence (e.g., by genetic engineering) to generate a chimeric nucleotide sequence encoding a chimeric polypeptide.
  • a chimeric Cas9 polypeptide is generated by fusing a Cas9 polypeptide (e.g., wild type Cas9 or a Cas9 variant, e.g., a Cas9 with reduced or inactivated nuclease activity) with a heterologous sequence that provides for subcellular localization (e.g., a nuclear localization signal (NLS) for targeting to the nucleus; a mitochondrial localization signal for targeting to the mitochondria; a chloroplast localization signal for targeting to a chloroplast; an ER retention signal; and the like).
  • a nuclear localization signal NLS
  • the heterologous sequence can provide a tag for ease of tracking or purification (e.g., a fluorescent protein, e.g., green fluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato, and the like; a HIS tag, e.g., a 6 ⁇ His tag; a hemagglutinin (HA) tag; a FLAG tag; a Myc tag; and the like).
  • GFP green fluorescent protein
  • RFP red fluorescent protein
  • CFP CFP
  • mCherry mCherry
  • tdTomato e.g., a fluorescent protein
  • HIS tag e.g., a 6 ⁇ His tag
  • HA hemagglutinin
  • FLAG tag e.g., hemagglutinin
  • Myc tag e.g., Myc tag
  • the heterologous sequence can provide a binding domain (e.g., to provide the ability of a chimeric Cas9 polypeptide to bind to another protein of interest, e.g., a DNA or histone modifying protein, a transcription factor or transcription repressor, a recruiting protein, etc.).
  • a binding domain e.g., to provide the ability of a chimeric Cas9 polypeptide to bind to another protein of interest, e.g., a DNA or histone modifying protein, a transcription factor or transcription repressor, a recruiting protein, etc.
  • the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a chimeric site-directed modifying polypeptide.
  • the nucleic acid comprising a nucleotide sequence encoding a chimeric site-directed modifying polypeptide is an expression vector, e.g., a recombinant expression vector.
  • a method involves contacting a target DNA or introducing into a cell (or a population of cells) one or more nucleic acids comprising a chimeric site-directed modifying polypeptide.
  • Suitable nucleic acids comprising nucleotide sequences encoding a chimeric site-directed modifying polypeptide include expression vectors, where an expression vector comprising a nucleotide sequence encoding a chimeric site-directed modifying polypeptide is a “recombinant expression vector.”
  • the recombinant expression vector is a viral construct, e.g., a recombinant adeno-associated virus construct (see, e.g., U.S. Pat. No. 7,078,387), a recombinant adenoviral construct, a recombinant lentiviral construct, etc.
  • a viral construct e.g., a recombinant adeno-associated virus construct (see, e.g., U.S. Pat. No. 7,078,387), a recombinant adenoviral construct, a recombinant lentiviral construct, etc.
  • Suitable expression vectors include, but are not limited to, viral vectors (e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis
  • SV40 herpes simplex virus
  • human immunodeficiency virus see, e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816, 1999
  • a retroviral vector e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus
  • retroviral vector e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myelop
  • Suitable expression vectors are known to those of skill in the art, and many are commercially available.
  • the following vectors are provided by way of example; for eukaryotic host cells: pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia).
  • any other vector may be used so long as it is compatible with the host cell.
  • any of a number of suitable transcription and translation control elements including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector (see e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544).
  • a nucleotide sequence encoding a chimeric site-directed modifying polypeptide is operably linked to a control element, e.g., a transcriptional control element, such as a promoter.
  • a control element e.g., a transcriptional control element, such as a promoter.
  • the transcriptional control element may be functional in either a eukaryotic cell, e.g., a mammalian cell; or a prokaryotic cell (e.g., bacterial or archaeal cell).
  • a nucleotide sequence encoding a chimeric site-directed modifying polypeptide is operably linked to multiple control elements that allow expression of the nucleotide sequence encoding a chimeric site-directed modifying polypeptide in both prokaryotic and eukaryotic cells.
  • eukaryotic promoters include those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
  • the expression vector may also contain a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector may also include appropriate sequences for amplifying expression.
  • the expression vector may also include nucleotide sequences encoding protein tags (e.g., 6 ⁇ His tag, hemagglutinin (HA) tag, a fluorescent protein (e.g., a green fluorescent protein; a yellow fluorescent protein, etc.), etc.) that are fused to the chimeric site-directed modifying polypeptide.
  • protein tags e.g., 6 ⁇ His tag, hemagglutinin (HA) tag, a fluorescent protein (e.g., a green fluorescent protein; a yellow fluorescent protein, etc.
  • a nucleotide sequence encoding a chimeric site-directed modifying polypeptide is operably linked to an inducible promoter (e.g., heat shock promoter, Tetracycline-regulated promoter, Steroid-regulated promoter, Metal-regulated promoter, estrogen receptor-regulated promoter, etc.).
  • a nucleotide sequence encoding a chimeric site-directed modifying polypeptide is operably linked to a spatially restricted and/or temporally restricted promoter (e.g., a tissue specific promoter, a cell type specific promoter, etc.).
  • a nucleotide sequence encoding a chimeric site-directed modifying polypeptide is operably linked to a constitutive promoter.
  • nucleic acid e.g., an expression construct
  • Suitable methods include, include e.g., viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, nanoparticle-mediated nucleic acid delivery (see, e.g., Panyam et., al Adv Drug Deliv Rev. 2012 Sep. 13. pii: 50169-409X(12)00283-9. doi: 10.1016/j.addr.2012.09.023), and the like.
  • PKI polyethyleneimine
  • a method involves contacting a target DNA with a complex (a “targeting complex”), which complex comprises a guide RNA and a site-directed modifying polypeptide.
  • a guide RNA and a site-directed modifying polypeptide form a complex.
  • the guide RNA provides target specificity to the complex by comprising a nucleotide sequence that is complementary to a sequence of a target DNA.
  • the site-directed modifying polypeptide of the complex provides the site-specific activity.
  • a complex modifies a target DNA, leading to, for example, DNA cleavage, DNA methylation, DNA damage, DNA repair, etc.
  • a complex modifies a target polypeptide associated with target DNA (e.g., a histone, a DNA-binding protein, etc.), leading to, for example, histone methylation, histone acetylation, histone ubiquitination, and the like.
  • target DNA e.g., a histone, a DNA-binding protein, etc.
  • the target DNA may be, for example, naked DNA in vitro, chromosomal DNA in cells in vitro, chromosomal DNA in cells in vivo, etc.
  • the site-directed modifying polypeptide exhibits nuclease activity that cleaves target DNA at a target DNA sequence defined by the region of complementarity between the guide RNA and the target DNA.
  • site-directed modifying polypeptide is a Cas9 or Cas9 related polypeptide
  • site-specific cleavage of the target DNA occurs at locations determined by both (i) base-pairing complementarity between the guide RNA and the target DNA; and (ii) a short motif [referred to as the protospacer adjacent motif (PAM)] in the target DNA.
  • PAM protospacer adjacent motif
  • the PAM sequence of the non-complementary strand is 5′-XGG-3′, where X is any DNA nucleotide and X is immediately 3′ of the target sequence of the non-complementary strand of the target DNA.
  • the PAM sequence of the complementary strand is 5′-CCY-3′, where Y is any DNA nucleotide and Y is immediately 5′ of the target sequence of the complementary strand of the target DNA (where the PAM of the non-complementary strand is 5′-GGG-3′ and the PAM of the complementary strand is 5′-CCC-3′).
  • different Cas9 proteins may be advantageous to use in the various provided methods in order to capitalize on various enzymatic characteristics of the different Cas9 proteins (e.g., for different PAM sequence preferences; for increased or decreased enzymatic activity; for an increased or decreased level of cellular toxicity; to change the balance between NHEJ, homology-directed repair, single strand breaks, double strand breaks, etc.).
  • Cas9 proteins from various species may require different PAM sequences in the target DNA.
  • the PAM sequence requirement may be different than the 5′-XGG-3′ sequence described above.
  • the present disclosure provides a C. jejuni PAM sequence NNNNACA; P. multocida PAM sequences GNNNCNNA or NNNNC; an F. novicida PAM sequence NG; an S. thermophilus ** PAM sequence NNAAAAW; an L. innocua PAM sequence NGG; and an S. dysgalactiae PAM sequence NGG.
  • Exemplary methods provided that take advantage of characteristics of Cas9 orthologs include the following.
  • a method for manipulating DNA in a cell comprising contacting the DNA with a Cas9 ortholog-guideRNA complex, wherein the complex comprises: (a) a cognate guide RNA for a first Cas9 endonuclease from a cluster in Supplementary Table S2 and (b) a second Cas9 endonuclease from the cluster that is exchangeable with preserved high cleavage efficiency with the first endonuclease and shares at least 80% identity with the first endonuclease over 80% of their length.
  • the guide is a single-molecule guide RNA.
  • the guide RNA is a double-molecule guide RNA.
  • the first Cas9 endonuclease is from S. pyogenes and the second Cas9 endonuclease is from S. mutans .
  • the first Cas9 endonuclease is from S. theromophilus * and the second Cas9 endonuclease is from S. mutans .
  • the first Cas9 endonuclease is from N. meningitidis and the second Cas9 endonuclease is from P. multocida.
  • a method for manipulating DNA in a cell comprising contacting the DNA with a Cas9 ortholog-guideRNA complex, wherein the complex comprises: (a) a cognate guide RNA of a first Cas9 endonuclease from a cluster in Supplementary Table S6 and (b) an Cas9 endonuclease from a cluster in Supplementary Table S6 that is exchangeable with lowered cleavage efficiency with the first endonuclease and shares at least 50% amino acid sequence identity with the first endonuclease over 70% of their length.
  • the guide is a single-molecule guide RNA.
  • the guide RNA is a double-molecule guide RNA.
  • the first Cas9 endonuclease is from C. Jejuni and the second Cas9 endonuclease is from P. multocida .
  • the first Cas9 endonuclease is from N. meningitidis and the second Cas9 endonuclease is from P. multocida.
  • a method for manipulating DNA in a cell comprising contacting the DNA with two or more Cas9-guideRNA complexes, wherein each Cas9-guideRNA complex comprises: (a) a Cas9 endonuclease from a different cluster in Supplementary Table S6 exhibiting less than 50% amino acid sequence identity with the other endonucleases of the method over 70% of their length, and (b) a guide RNA specifically complexed with each Cas9 endonuclease.
  • the guide is a single-molecule guide RNA.
  • the guide RNA is a double-molecule guide RNA.
  • the Cas9 endonucleases are from F. novicida and S.
  • the Cas9 endonucleases are from N. meningitidis and S. mutans .
  • Cas9 orthologs from a wide variety of species have been identified herein. All identified Cas9 orthologs have the same domain architecture with a central HNH endonuclease domain and a split RuvC/RNaseH domain. Cas9 proteins share four key motifs with a conserved architecture. Motifs 1, 2, and 4 are RuvC like motifs while motif 3 is an HNH-motif.
  • a suitable site-directed modifying polypeptide comprises an amino acid sequence having four motifs, each of motifs 1-4 having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100% amino acid sequence identity to the motifs 1-4 of the Cas9 amino acid sequence depicted in Table 1), or to the corresponding portions in any of the amino acid sequences set forth in SEQ ID NOs: 1-800.
  • a suitable site-directed modifying polypeptide comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100% amino acid sequence identity to amino acids 7-166 and/or 731-1003 of SEQ ID NO: 8, or to the corresponding portions in any of the amino acid sequences set forth as SEQ ID NOs: 1-800.
  • the nuclease activity cleaves target DNA to produce double strand breaks. These breaks are then repaired by the cell in one of two ways: non-homologous end joining, and homology-directed repair.
  • non-homologous end joining NHEJ
  • homology-directed repair a donor polynucleotide with homology to the cleaved target DNA sequence is used as a template for repair of the cleaved target DNA sequence, resulting in the transfer of genetic information from the donor polynucleotide to the target DNA.
  • new nucleic acid material may be inserted/copied into the site.
  • a target DNA is contacted with a donor polynucleotide.
  • a donor polynucleotide is introduced into a cell.
  • the modifications of the target DNA due to NHEJ and/or homology-directed repair lead to, for example, gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, gene disruption, gene mutation, sequence replacement, etc.
  • cleavage of DNA by a site-directed modifying polypeptide may be used to delete nucleic acid material from a target DNA sequence (e.g., to disrupt a gene that makes cells susceptible to infection (e.g.
  • the methods can be used to knock out a gene (resulting in complete lack of transcription or altered transcription) or to knock in genetic material into a locus of choice in the target DNA.
  • RNA and a site-directed modifying polypeptide are coadministered to cells with a donor polynucleotide sequence that includes at least a segment with homology to the target DNA sequence
  • the subject methods may be used to add, i.e. insert or replace, nucleic acid material to a target DNA sequence (e.g.
  • a tag e.g., 6 ⁇ His, a fluorescent protein (e.g., a green fluorescent protein; a yellow fluorescent protein, etc.), hemagglutinin (HA), FLAG, etc.
  • a regulatory sequence e.g. promoter, polyadenylation signal, internal ribosome entry sequence (IRES), 2A peptide, start codon, stop codon, splice signal, localization signal, etc.
  • a nucleic acid sequence e.g., introduce a mutation
  • a complex comprising a guide RNA and a site-directed modifying polypeptide is useful in any in vitro or in vivo application in which it is desirable to modify DNA in a site-specific, i.e. “targeted”, way, for example gene knock-out, gene knock-in, gene editing, gene tagging, sequence replacement, etc., as used in, for example, gene therapy, e.g. to treat a disease or as an antiviral, antipathogenic, or anticancer therapeutic, the production of genetically modified organisms in agriculture, the large scale production of proteins by cells for therapeutic, diagnostic, or research purposes, the induction of iPS cells, biological research, the targeting of genes of pathogens for deletion or replacement, etc.
  • a site-specific i.e. “targeted”
  • gene therapy e.g. to treat a disease or as an antiviral, antipathogenic, or anticancer therapeutic
  • the production of genetically modified organisms in agriculture the large scale production of proteins by cells for therapeutic, diagnostic, or research purposes
  • the site-directed modifying polypeptide comprises a modified form of the Cas9 protein.
  • the modified form of the Cas9 protein comprises an amino acid change (e.g., deletion, insertion, or substitution) that reduces the naturally-occurring nuclease activity of the Cas9 protein.
  • the modified form of the Cas9 protein has less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nuclease activity of the corresponding wild-type Cas9 polypeptide.
  • the modified form of the Cas9 polypeptide has no substantial nuclease activity.
  • dCas9 When a site-directed modifying polypeptide is a modified form of the Cas9 polypeptide that has no substantial nuclease activity, it can be referred to as “dCas9.”
  • the modified form of the Cas9 polypeptide is a D10A (aspartate to alanine at amino acid position 10 of SEQ ID NO:8) mutation (or the corresponding mutation of any of the proteins set forth as SEQ ID NOs: 1-800) that can cleave the complementary strand of the target DNA but has reduced ability to cleave the non-complementary strand of the target DNA (thus resulting in a single strand break (SSB) instead of a DSB).
  • D10A aspartate to alanine at amino acid position 10 of SEQ ID NO:8 mutation (or the corresponding mutation of any of the proteins set forth as SEQ ID NOs: 1-800) that can cleave the complementary strand of the target DNA but has reduced ability to cleave the non-complementary strand of the target DNA (thus resulting in a single strand break (SSB) instead of a DSB).
  • SSB single strand break
  • the modified form of the Cas9 polypeptide is a H840A (histidine to alanine at amino acid position 840 of SEQ ID NO:8) mutation (or the corresponding mutation of any of the proteins set forth as SEQ ID NOs: 1-800) that can cleave the non-complementary strand of the target DNA but has reduced ability to cleave the complementary strand of the target DNA (thus resulting in a single strand break (SSB) instead of a DSB).
  • H840A histidine to alanine at amino acid position 840 of SEQ ID NO:8 mutation
  • SEQ ID NOs: 1-800 the corresponding mutation of any of the proteins set forth as SEQ ID NOs: 1-800
  • D10A or H840A variant of SEQ ID NO: 8 Cas9 can alter the expected biological outcome because the non-homologous end joining (NHEJ) is much more likely to occur when DSBs are present as opposed to SSBs.
  • NHEJ non-homologous end joining
  • a D10A or H840A variant of Cas9 can be used.
  • Other residues can be mutated to achieve the same effect (i.e. inactivate one or the other nuclease portions).
  • pyogenes Cas9 residues D10, G12, G17, E762, H840, N863, H982, H983, A984, D986, and/or A987 can be altered (i.e., substituted). Also, mutations other than alanine substitutions are contemplated.
  • a site-directed polypeptide e.g., site-directed modifying polypeptide
  • a SEQ ID NO: 8 Cas9 protein has a D10, G12, G17, E762, H840, N863, H982, H983, A984, D986, and/or a A987 mutation, e.g., D 10A, G12A, G17A, E762A, H840A, N863A, H982A, H983A, A984A, and/or D986A
  • the polypeptide can still bind to target DNA in a site-specific manner (because it is still guided to a target DNA sequence by a guide RNA) as long as it retains the ability to interact with the guide RNA.
  • the modified form of the SEQ ID NO: 8 Cas9 polypeptide harbors both the D10A and the H840A mutations (or the corresponding mutations of any of the proteins set forth as SEQ ID NOs: 1-800) such that the polypeptide has a reduced ability to cleave both the complementary and the non-complementary strands of the target DNA (i.e., the variant can have no substantial nuclease activity).
  • Other residues can be mutated to achieve the same effect (i.e. inactivate one or the other nuclease portions).
  • SEQ ID NO: 8 residues D10, G12, G17, E762, H840, N863, H982, H983, A984, D986, and/or A987 can be altered (i.e., substituted). Also, mutations other than alanine substitutions are contemplated.
  • the site-directed modifying polypeptide comprises a heterologous sequence (e.g., a fusion).
  • a heterologous sequence can provide for subcellular localization of the site-directed modifying polypeptide (e.g., a nuclear localization signal (NLS) for targeting to the nucleus; a mitochondrial localization signal for targeting to the mitochondria; a chloroplast localization signal for targeting to a chloroplast; a ER retention signal; and the like).
  • NLS nuclear localization signal
  • a heterologous sequence can provide a tag for ease of tracking or purification (e.g., a fluorescent protein, e.g., green fluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato, and the like; a his tag, e.g., a 6 ⁇ His tag; a hemagglutinin (HA) tag; a FLAG tag; a Myc tag; and the like).
  • the heterologous sequence can provide for increased or decreased stability.
  • a site-directed modifying polypeptide can be codon-optimized. This type of optimization is known in the art and entails the mutation of foreign-derived DNA to mimic the codon preferences of the intended host organism or cell while encoding the same protein. Thus, the codons are changed, but the encoded protein remains unchanged.
  • a human codon-optimized Cas9 or variant, e.g., enzymatically inactive variant
  • Any suitable site-directed modifying polypeptide e.g., any Cas9 such as any of the sequences set forth in SEQ ID NOs: 1-800
  • a mouse codon-optimized Cas9 or variant, e.g., enzymatically inactive variant
  • codon optimization is not required, it is acceptable and may be preferable in certain cases.
  • Polyadenylation signals can also be chosen to optimize expression in the intended host.
  • a guide RNA and a site-directed modifying polypeptide are used as an inducible system for shutting off gene expression in bacterial cells.
  • nucleic acids encoding an appropriate guide RNA and/or an appropriate site-directed polypeptide are incorporated into the chromosome of a target cell and are under control of an inducible promoter.
  • the target DNA is cleaved (or otherwise modified) at the location of interest (e.g., a target gene on a separate plasmid), when both the guide RNA and the site-directed modifying polypeptide are present and form a complex.
  • bacterial expression strains are engineered to include nucleic acid sequences encoding an appropriate site-directed modifying polypeptide in the bacterial genome and/or an appropriate guide RNA on a plasmid (e.g., under control of an inducible promoter), allowing experiments in which the expression of any targeted gene (expressed from a separate plasmid introduced into the strain) could be controlled by inducing expression of the guide RNA and the site-directed polypeptide.
  • the site-directed modifying polypeptide has enzymatic activity that modifies target DNA in ways other than introducing double strand breaks.
  • Enzymatic activity of interest that may be used to modify target DNA (e.g., by fusing a heterologous polypeptide with enzymatic activity to a site-directed modifying polypeptide, thereby generating a chimeric site-directed modifying polypeptide) includes, but is not limited methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylase activity). Methylation and demethylation is recognized in the art as an important mode of epigenetic gene regulation while DNA damage and repair activity is essential for
  • the methods herein find use in the epigenetic modification of target DNA and may be employed to control epigenetic modification of target DNA at any location in a target DNA by genetically engineering the desired complementary nucleic acid sequence into the DNA-targeting segment of a guide RNA.
  • the methods herein also find use in the intentional and controlled damage of DNA at any desired location within the target DNA.
  • the methods herein also find use in the sequence-specific and controlled repair of DNA at any desired location within the target DNA. Methods to target DNA-modifying enzymatic activities to specific locations in target DNA find use in both research and clinical applications.
  • the site-directed modifying polypeptide has activity that modulates the transcription of target DNA (e.g., in the case of a chimeric site-directed modifying polypeptide, etc.).
  • a chimeric site-directed modifying polypeptides comprising a heterologous polypeptide that exhibits the ability to increase or decrease transcription (e.g., transcriptional activator or transcription repressor polypeptides) is used to increase or decrease the transcription of target DNA at a specific location in a target DNA, which is guided by the DNA-targeting segment of the guide RNA.
  • source polypeptides for providing a chimeric site-directed modifying polypeptide with transcription modulatory activity include, but are not limited to light-inducible transcription regulators, small molecule/drug-responsive transcription regulators, transcription factors, transcription repressors, etc.
  • the method is used to control the expression of a targeted coding-RNA (protein-encoding gene) and/or a targeted non-coding RNA (e.g., tRNA, rRNA, snoRNA, siRNA, miRNA, long ncRNA, etc.).
  • the site-directed modifying polypeptide has enzymatic activity that modifies a polypeptide associated with DNA (e.g. histone).
  • the enzymatic activity is methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity (i.e., ubiquitination activity), deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, demyristoylation activity glycosylation activity (e.g., from GlcNAc transferase) or deglycosylation activity.
  • ubiquitin ligase activity i.e., ubiquitination activity
  • deubiquitinating activity i.e., ubiquitinating activity
  • adenylation activity deadenylation activity
  • SUMOylating activity deSUMOylating activity
  • deSUMOylating activity de
  • the enzymatic activities listed herein catalyze covalent modifications to proteins. Such modifications are known in the art to alter the stability or activity of the target protein (e.g., phosphorylation due to kinase activity can stimulate or silence protein activity depending on the target protein). Of particular interest as protein targets are histones. Histone proteins are known in the art to bind DNA and form complexes known as nucleosomes. Histones can be modified (e.g., by methylation, acetylation, ubuitination, phosphorylation) to elicit structural changes in the surrounding DNA, thus controlling the accessibility of potentially large portions of DNA to interacting factors such as transcription factors, polymerases and the like.
  • a single histone can be modified in many different ways and in many different combinations (e.g., trimethylation of lysine 27 of histone 3, H3K27, is associated with DNA regions of repressed transcription while trimethylation of lysine 4 of histone 3, H3K4, is associated with DNA regions of active transcription).
  • a site-directed modifying polypeptide with histone-modifying activity finds use in the site specific control of DNA structure and can be used to alter the histone modification pattern in a selected region of target DNA. Such methods find use in both research and clinical applications.
  • multiple guide RNAs are used simultaneously to simultaneously modify different locations on the same target DNA or on different target DNAs.
  • two or more guide RNAs target the same gene or transcript or locus.
  • two or more guide RNAs target different unrelated loci.
  • two or more guide RNAs target different, but related loci.
  • the site-directed modifying polypeptide is provided directly as a protein.
  • fungi e.g., yeast
  • spheroplast transformation see Kawai et al., Bioeng Bugs. 2010 November-December; 1(6):395-403: “Transformation of Saccharomyces cerevisiae and other fungi: methods and possible underlying mechanism”; and Tanka et al., Nature. 2004 March 18; 428(6980):323-8: “Conformational variations in an infectious protein determine prion strain differences”; both of which are herein incorporated by reference in their entirety).
  • a site-directed modifying polypeptide e.g., Cas9
  • a spheroplast with or without nucleic acid encoding a guide RNA and with or without a donor polynucleotide
  • the spheroplast can be used to introduce the content into a yeast cell.
  • a site-directed modifying polypeptide can be introduced into a cell (provided to the cell) by any convenient method; such methods are known to those of ordinary skill in the art.
  • a site-directed modifying polypeptide can be injected directly into a cell (e.g., with or without nucleic acid encoding a guide RNA and with or without a donor polynucleotide), e.g., a cell of a zebrafish embryo, the pronucleus of a fertilized mouse oocyte, etc.
  • a cell e.g., with or without nucleic acid encoding a guide RNA and with or without a donor polynucleotide
  • a cell of a zebrafish embryo e.g., a cell of a zebrafish embryo, the pronucleus of a fertilized mouse oocyte, etc.
  • the methods may be employed to induce DNA cleavage, DNA modification, and/or transcriptional modulation in mitotic or post-mitotic cells in vivo and/or ex vivo and/or in vitro (e.g., to produce genetically modified cells that can be reintroduced into an individual).
  • a mitotic and/or post-mitotic cell of interest in the disclosed methods may include a cell from any organism (e.g.
  • a bacterial cell e.g., a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a plant cell, an algal cell, e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C. Agardh , and the like, a fungal cell (e.g., a yeast cell), an animal cell, a cell from an invertebrate animal (e.g.
  • a cell from a vertebrate animal e.g., fish, amphibian, reptile, bird, mammal
  • a cell from a mammal e.g., a cell from a rodent, a cell from a primate, a cell from a human, etc.
  • a stem cell e.g. an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell, a germ cell; a somatic cell, e.g. a fibroblast, a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell; an in vitro or in vivo embryonic cell of an embryo at any stage, e.g., a 1-cell, 2-cell, 4-cell, 8-cell, etc. stage zebrafish embryo; etc.).
  • ES embryonic stem
  • iPS induced pluripotent stem
  • a germ cell e.g. a somatic cell, e.g. a fibroblast, a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell
  • an in vitro or in vivo embryonic cell of an embryo at any stage e
  • Cells may be from established cell lines or they may be primary cells, where “primary cells”, “primary cell lines”, and “primary cultures” are used interchangeably herein to refer to cells and cells cultures that have been derived from a and allowed to grow in vitro for a limited number of passages, i.e. splittings, of the culture.
  • primary cultures are cultures that may have been passaged 0 times, 1 time, 2 times, 4 times, 5 times, 10 times, or 15 times, but not enough times go through the crisis stage.
  • the primary cell lines of the present invention are maintained for fewer than 10 passages in vitro.
  • Target cells are in many embodiments unicellular organisms, or are grown in culture.
  • the cells may be harvest from an individual by any convenient method.
  • leukocytes may be conveniently harvested by apheresis, leukocytapheresis, density gradient separation, etc., while cells from tissues such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach, etc. are most conveniently harvested by biopsy.
  • An appropriate solution may be used for dispersion or suspension of the harvested cells.
  • Such solution will generally be a balanced salt solution, e.g.
  • fetal calf serum or other naturally occurring factors in conjunction with an acceptable buffer at low concentration, generally from 5-25 mM.
  • Convenient buffers include HEPES, phosphate buffers, lactate buffers, etc.
  • the cells may be used immediately, or they may be stored, frozen, for long periods of time, being thawed and capable of being reused.
  • the cells will usually be frozen in 10% DMSO, 50% serum, 40% buffered medium, or some other such solution as is commonly used in the art to preserve cells at such freezing temperatures, and thawed in a manner as commonly known in the art for thawing frozen cultured cells.
  • a method involves contacting a target DNA or introducing into a cell (or a population of cells) one or more nucleic acids comprising nucleotide sequences encoding a guide RNA and/or a site-directed modifying polypeptide and/or a donor polynucleotide.
  • Suitable nucleic acids comprising nucleotide sequences encoding a guide RNA and/or a site-directed modifying polypeptide include expression vectors, where an expression vector comprising a nucleotide sequence encoding a guide RNA and/or a site-directed modifying polypeptide is a “recombinant expression vector.”
  • the recombinant expression vector is a viral construct, e.g., a recombinant adeno-associated virus construct (see, e.g., U.S. Pat. No. 7,078,387), a recombinant adenoviral construct, a recombinant lentiviral construct, etc.
  • a viral construct e.g., a recombinant adeno-associated virus construct (see, e.g., U.S. Pat. No. 7,078,387), a recombinant adenoviral construct, a recombinant lentiviral construct, etc.
  • Suitable expression vectors include, but are not limited to, viral vectors (e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al, Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci
  • SV40 herpes simplex virus
  • human immunodeficiency virus see, e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et at, J Virol 73:7812 7816, 1999
  • a retroviral vector e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus
  • retroviral vector e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloprol
  • Suitable expression vectors are known to those of skill in the art, and many are commercially available.
  • the following vectors are provided by way of example; for eukaryotic host cells: pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia).
  • any other vector may be used so long as it is compatible with the host cell.
  • a nucleotide sequence encoding a guide RNA and/or a site-directed modifying polypeptide is operably linked to a control element, e.g., a transcriptional control element, such as a promoter.
  • a control element e.g., a transcriptional control element, such as a promoter.
  • the transcriptional control element may be functional in either a eukaryotic cell, e.g., a mammalian cell, or a prokaryotic cell (e.g., bacterial or archaeal cell).
  • a nucleotide sequence encoding a guide RNA and/or a site-directed modifying polypeptide is operably linked to multiple control elements that allow expression of the nucleotide sequence encoding a guide RNA and/or a site-directed modifying polypeptide in both prokaryotic and eukaryotic cells.
  • any of a number of suitable transcription and translation control elements including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector (e.g., U6 promoter, H1 promoter, etc.; see above) (see e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544).
  • a guide RNA and/or a site-directed modifying polypeptide can be provided as RNA.
  • the guide RNA and/or the RNA encoding the site-directed modifying polypeptide can be produced by direct chemical synthesis or may be transcribed in vitro from a DNA encoding the guide RNA. Methods of synthesizing RNA from a DNA template are well known in the art.
  • the guide RNA and/or the RNA encoding the site-directed modifying polypeptide will be synthesized in vitro using an RNA polymerase enzyme (e.g., T7 polymerase, T3 polymerase, SP6 polymerase, etc.). Once synthesized, the RNA may directly contact a target DNA or may be introduced into a cell by any of the well-known techniques for introducing nucleic acids into cells (e.g., microinjection, electroporation, transfection, etc).
  • Nucleotides encoding a guide RNA (introduced either as DNA or RNA) and/or a site-directed modifying polypeptide (introduced as DNA or RNA) and/or a donor polynucleotide may be provided to the cells using well-developed transfection techniques; see, e.g. Angel and Yanik (2010) PLoS ONE 5(7): e 11756, and the commercially available TransMessenger® reagents from Qiagen, StemfectTM RNA Transfection Kit from Stemgent, and TransIT®-mRNA Transfection Kit from Mims Bio LLC. See also Beumer et al. (2008) Efficient gene targeting in Drosophila by direct embryo injection with zinc-finger nucleases.
  • nucleic acids encoding a guide RNA and/or a site-directed modifying polypeptide and/or a chimeric site-directed modifying polypeptide and/or a donor polynucleotide may be provided on DNA vectors.
  • Many vectors, e.g. plasmids, cosmids, minicircles, phage, viruses, etc., useful for transferring nucleic acids into target cells are available.
  • the vectors comprising the nucleic acid(s) may be maintained episomally, e.g.
  • plasmids as plasmids, minicircle DNAs, viruses such cytomegalovirus, adenovirus, etc., or they may be integrated into the target cell genome, through homologous recombination or random integration, e.g. retrovirus-derived vectors such as MMLV, HIV-1, ALV, etc.
  • Vectors may be provided directly to the cells.
  • the cells are contacted with vectors comprising the nucleic acid encoding guide RNA and/or a site-directed modifying polypeptide and/or a chimeric site-directed modifying polypeptide and/or a donor polynucleotide such that the vectors are taken up by the cells.
  • Methods for contacting cells with nucleic acid vectors that are plasmids including electroporation, calcium chloride transfection, microinjection, and lipofection are well known in the art.
  • the cells are contacted with viral particles comprising the nucleic acid encoding a guide RNA and/or a site-directed modifying polypeptide and/or a chimeric site-directed modifying polypeptide and/or a donor polynucleotide.
  • Retroviruses for example, lentiviruses, are particularly suitable to the method of the invention. Commonly used retroviral vectors are “defective”, i.e. unable to produce viral proteins required for productive infection. Rather, replication of the vector requires growth in a packaging cell line. To generate viral particles comprising nucleic acids of interest, the retroviral nucleic acids comprising the nucleic acid are packaged into viral capsids by a packaging cell line.
  • Different packaging cell lines provide a different envelope protein (ecotropic, amphotropic or xenotropic) to be incorporated into the capsid, this envelope protein determining the specificity of the viral particle for the cells (ecotropic for murine and rat; amphotropic for most mammalian cell types including human, dog and mouse; and xenotropic for most mammalian cell types except murine cells).
  • the appropriate packaging cell line may be used to ensure that the cells are targeted by the packaged viral particles.
  • Methods of introducing the retroviral vectors comprising the nucleic acid encoding the reprogramming factors into packaging cell lines and of collecting the viral particles that are generated by the packaging lines are well known in the art. Nucleic acids can also introduced by direct micro-injection (e.g., injection of RNA into a zebrafish embryo).
  • Vectors used for providing the nucleic acids encoding guide RNA and/or a site-directed modifying polypeptide and/or a chimeric site-directed modifying polypeptide and/or a donor polynucleotide to the cells will typically comprise suitable promoters for driving the expression, that is, transcriptional activation, of the nucleic acid of interest.
  • the nucleic acid of interest will be operably linked to a promoter. This may include ubiquitously acting promoters, for example, the CMV-13-actin promoter, or inducible promoters, such as promoters that are active in particular cell populations or that respond to the presence of drugs such as tetracycline.
  • vectors used for providing a guide RNA and/or a site-directed modifying polypeptide and/or a chimeric site-directed modifying polypeptide and/or a donor polynucleotide to the cells may include nucleic acid sequences that encode for selectable markers in the target cells, so as to identify cells that have taken up the guide RNA and/or a site-directed modifying polypeptide and/or a chimeric site-directed modifying polypeptide and/or a donor polynucleotide.
  • a guide RNA and/or a site-directed modifying polypeptide and/or a chimeric site-directed modifying polypeptide may instead be used to contact DNA or introduced into cells as RNA.
  • Methods of introducing RNA into cells are known in the art and may include, for example, direct injection, transfection, or any other method used for the introduction of DNA.
  • a site-directed modifying polypeptide may instead be provided to cells as a polypeptide.
  • Such a polypeptide may optionally be fused to a polypeptide domain that increases solubility of the product. The domain may be linked to the polypeptide through a defined protease cleavage site, e.g. a TEV sequence, which is cleaved by TEV protease.
  • the linker may also include one or more flexible sequences, e.g. from 1 to 10 glycine residues.
  • the cleavage of the fusion protein is performed in a buffer that maintains solubility of the product, e.g. in the presence of from 0.5 to 2 M urea, in the presence of polypeptides and/or polynucleotides that increase solubility, and the like.
  • Domains of interest include endosomolytic domains, e.g. influenza HA domain; and other polypeptides that aid in production, e.g. IF2 domain, GST domain, GRPE domain, and the like.
  • the polypeptide may be formulated for improved stability.
  • the peptides may be PEGylated, where the polyethyleneoxy group provides for enhanced lifetime in the blood stream.
  • the site-directed modifying polypeptide may be fused to a polypeptide permeant domain to promote uptake by the cell.
  • permeant domains are known in the art and may be used in the non-integrating polypeptides of the present invention, including peptides, peptidomimetics, and non-peptide carriers.
  • a permeant peptide may be derived from the third alpha helix of Drosophila melanogaster transcription factor Antennapaedia, referred to as penetratin, which comprises the amino acid sequence RQIKIWFQNRRMKWKK.
  • the permeant peptide comprises the HIV-1 tat basic region amino acid sequence, which may include, for example, amino acids 49-57 of naturally-occurring tat protein.
  • Other permeant domains include polyarginine motifs, for example, the region of amino acids 34-56 of HIV-1 rev protein, nona-arginine, octa-arginine, and the like.
  • the nona-arginine (R9) sequence is one of the more efficient PTDs that have been characterized (Wender et al. 2000; Uemura et al. 2002).
  • the site at which the fusion is made may be selected in order to optimize the biological activity, secretion or binding characteristics of the polypeptide. The optimal site will be determined by routine experimentation.
  • a site-directed modifying polypeptide may be produced in vitro or by eukaryotic cells or by prokaryotic cells, and it may be further processed by unfolding, e.g. heat denaturation, DTT reduction, etc. and may be further refolded, using methods known in the art.
  • Modifications of interest that do not alter primary sequence include chemical derivatization of polypeptides, e.g., acylation, acetylation, carboxylation, amidation, etc. Also included are modifications of glycosylation, e.g. those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g. by exposing the polypeptide to enzymes which affect glycosylation, such as mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences that have phosphorylated amino acid residues, e.g. phosphotyrosine, phosphoserine, or phosphothreonine.
  • modifications of glycosylation e.g. those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g. by exposing the polypeptide to enzymes which affect glycosylation, such as mammalian glycosylating or
  • guide RNAs and site-directed modifying polypeptides that have been modified using ordinary molecular biological techniques and synthetic chemistry so as to improve their resistance to proteolytic degradation, to change the target sequence specificity, to optimize solubility properties, to alter protein activity (e.g., transcription modulatory activity, enzymatic activity, etc) or to render them more suitable as a therapeutic agent.
  • Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g. D-amino acids or non-naturally occurring synthetic amino acids. D-amino acids may be substituted for some or all of the amino acid residues.
  • the site-directed modifying polypeptides may be prepared by in vitro synthesis, using conventional methods as known in the art.
  • Various commercial synthetic apparatuses are available, for example, automated synthesizers by Applied Biosystems, Inc., Beckman, etc. By using synthesizers, naturally occurring amino acids may be substituted with unnatural amino acids. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like.
  • cysteines can be used to make thioethers, histidines for linking to a metal ion complex, carboxyl groups for forming amides or esters, amino groups for forming amides, and the like.
  • the site-directed modifying polypeptides may also be isolated and purified in accordance with conventional methods of recombinant synthesis.
  • a lysate may be prepared of the expression host and the lysate purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique.
  • the compositions which are used will comprise at least 20% by weight of the desired product, more usually at least about 75% by weight, preferably at least about 95% by weight, and for therapeutic purposes, usually at least about 99.5% by weight, in relation to contaminants related to the method of preparation of the product and its purification. Usually, the percentages will be based upon total protein.
  • the guide RNA and/or the site-directed modifying polypeptide and/or the donor polynucleotide are provided to the cells for about 30 minutes to about 24 hours, e.g., 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 18 hours, 20 hours, or any other period from about 30 minutes to about 24 hours, which may be repeated with a frequency of about every day to about every 4 days, e.g., every 1.5 days, every 2 days, every 3 days, or any other frequency from about every day to about every four days.
  • the agent(s) may be provided to the cells one or more times, e.g. one time, twice, three times, or more than three times, and the cells allowed to incubate with the agent(s) for some amount of time following each contacting event e.g. 16-24 hours, after which time the media is replaced with fresh media and the cells are cultured further.
  • the complexes may be provided simultaneously (e.g. as two polypeptides and/or nucleic acids), or delivered simultaneously. Alternatively, they may be provided consecutively, e.g. the targeting complex being provided first, followed by the second targeting complex, etc. or vice versa.
  • an effective amount of the guide RNA and/or site-directed modifying polypeptide and/or donor polynucleotide is provided to the target DNA or cells to induce target modification.
  • An effective amount of the guide RNA and/or site-directed modifying polypeptide and/or donor polynucleotide is the amount to induce a 2-fold increase or more in the amount of target modification observed between two homologous sequences relative to a negative control, e.g. a cell contacted with an empty vector or irrelevant polypeptide.
  • an effective amount or dose of the guide RNA and/or site-directed modifying polypeptide and/or donor polynucleotide will induce a 2-fold increase, a 3-fold increase, a 4-fold increase or more in the amount of target modification observed at a target DNA region, in some instances a 5-fold increase, a 6-fold increase or more, sometimes a 7-fold or 8-fold increase or more in the amount of recombination observed, e.g. an increase of 10-fold, 50-fold, or 100-fold or more, in some instances, an increase of 200-fold, 500-fold, 700-fold, or 1000-fold or more, e.g. a 5000-fold, or 10,000-fold increase in the amount of recombination observed.
  • the amount of target modification may be measured by any convenient method.
  • a silent reporter construct comprising complementary sequence to the targeting segment (targeting sequence) of the guide RNA flanked by repeat sequences that, when recombined, will reconstitute a nucleic acid encoding an active reporter may be cotransfected into the cells, and the amount of reporter protein assessed after contact with the guide RNA and/or site-directed modifying polypeptide and/or donor polynucleotide, e.g. 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours or more after contact with the guide RNA and/or site-directed modifying polypeptide and/or donor polynucleotide.
  • the extent of recombination at a genomic DNA region of interest comprising target DNA sequences may be assessed by PCR or Southern hybridization of the region after contact with a guide RNA and/or site-directed modifying polypeptide and/or donor polynucleotide, e.g. 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours or more after contact with the guide RNA and/or site-directed modifying polypeptide and/or donor polynucleotide.
  • a guide RNA and/or site-directed modifying polypeptide and/or donor polynucleotide may occur in any culture media and under any culture conditions that promote the survival of the cells.
  • cells may be suspended in any appropriate nutrient medium that is convenient, such as Iscove's modified DMEM or RPMI 1640, supplemented with fetal calf serum or heat inactivated goat serum (about 5-10%), L-glutamine, a thiol, particularly 2-mercaptoethanol, and antibiotics, e.g. penicillin and streptomycin.
  • the culture may contain growth factors to which the cells are responsive.
  • Growth factors are molecules capable of promoting survival, growth and/or differentiation of cells, either in culture or in the intact tissue, through specific effects on a transmembrane receptor. Growth factors include polypeptides and non-polypeptide factors. Conditions that promote the survival of cells are typically permissive of nonhomologous end joining and homology-directed repair. In applications in which it is desirable to insert a polynucleotide sequence into a target DNA sequence, a polynucleotide comprising a donor sequence to be inserted is also provided to the cell.
  • donor sequence or “donor polynucleotide” it is meant a nucleic acid sequence to be inserted at the cleavage site induced by a site-directed modifying polypeptide.
  • the donor polynucleotide will contain sufficient homology to a genomic sequence at the cleavage site, e.g. 70%, 80%, 85%, 90%, 95%, or 100% homology with the nucleotide sequences flanking the cleavage site, e.g. within about 50 bases or less of the cleavage site, e.g.
  • Donor sequences can be of any length, e.g.
  • nucleotides or more 10 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 500 nucleotides or more, 1000 nucleotides or more, 5000 nucleotides or more, etc.
  • the donor sequence is typically not identical to the genomic sequence that it replaces. Rather, the donor sequence may contain at least one or more single base changes, insertions, deletions, inversions or rearrangements with respect to the genomic sequence, so long as sufficient homology is present to support homology-directed repair.
  • the donor sequence comprises a non-homologous sequence flanked by two regions of homology, such that homology-directed repair between the target DNA region and the two flanking sequences results in insertion of the non-homologous sequence at the target region.
  • Donor sequences may also comprise a vector backbone containing sequences that are not homologous to the DNA region of interest and that are not intended for insertion into the DNA region of interest.
  • the homologous region(s) of a donor sequence will have at least 50% sequence identity to a genomic sequence with which recombination is desired. In certain embodiments, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 99.9% sequence identity is present. Any value between 1% and 100% sequence identity can be present, depending upon the length of the donor polynucleotide.
  • the donor sequence may comprise certain sequence differences as compared to the genomic sequence, e.g.
  • restriction sites nucleotide polymorphisms, selectable markers (e.g., drug resistance genes, fluorescent proteins, enzymes etc.), etc., which may be used to assess for successful insertion of the donor sequence at the cleavage site or in some cases may be used for other purposes (e.g., to signify expression at the targeted genomic locus).
  • selectable markers e.g., drug resistance genes, fluorescent proteins, enzymes etc.
  • nucleotide sequence differences will not change the amino acid sequence, or will make silent amino acid changes (i.e., changes which do not affect the structure or function of the protein).
  • these sequences differences may include flanking recombination sequences such as FLPs, IoxP sequences, or the like, that can be activated at a later time for removal of the marker sequence.
  • the donor sequence may be provided to the cell as single-stranded DNA, single-stranded RNA, double-stranded DNA, or double-stranded RNA. It may be introduced into a cell in linear or circular form. If introduced in linear form, the ends of the donor sequence may be protected (e.g., from exonucleolytic degradation) by methods known to those of skill in the art. For example, one or more dideoxynucleotide residues are added to the 3′ terminus of a linear molecule and/or self-complementary oligonucleotides are ligated to one or both ends. See, for example, Chang et al. (1987) Proc. Natl.
  • Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group(s) and the use of modified internucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues.
  • additional lengths of sequence may be included outside of the regions of homology that can be degraded without impacting recombination.
  • a donor sequence can be introduced into a cell as part of a vector molecule having additional sequences such as, for example, replication origins, promoters and genes encoding antibiotic resistance.
  • donor sequences can be introduced as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or poloxamer, or can be delivered by viruses (e.g., adenovirus, AAV), as described above for nucleic acids encoding a guide RNA and/or site-directed modifying polypeptide and/or donor polynucleotide.
  • viruses e.g., adenovirus, AAV
  • a DNA region of interest may be cleaved and modified, i.e. “genetically modified”, ex vivo.
  • the population of cells may be enriched for those comprising the genetic modification by separating the genetically modified cells from the remaining population.
  • the “genetically modified” cells may make up only about 1% or more (e.g., 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 15% or more, or 20% or more) of the cellular population.
  • Separation of “genetically modified” cells may be achieved by any convenient separation technique appropriate for the selectable marker used. For example, if a fluorescent marker has been inserted, cells may be separated by fluorescence activated cell sorting, whereas if a cell surface marker has been inserted, cells may be separated from the heterogeneous population by affinity separation techniques, e.g. magnetic separation, affinity chromatography, “panning” with an affinity reagent attached to a solid matrix, or other convenient technique.
  • Techniques providing accurate separation include fluorescence activated cell sorters, which can have varying degrees of sophistication, such as multiple color channels, low angle and obtuse light scattering detecting channels, impedance channels, etc.
  • the cells may be selected against dead cells by employing dyes associated with dead cells (e.g. propidium iodide). Any technique may be employed which is not unduly detrimental to the viability of the genetically modified cells.
  • Cell compositions that are highly enriched for cells comprising modified DNA are achieved in this manner.
  • “highly enriched” it is meant that the genetically modified cells will be 70% or more, 75% or more, 80% or more, 85% or more, 90% or more of the cell composition, for example, about 95% or more, or 98% or more of the cell composition.
  • the composition may be a substantially pure composition of genetically modified cells.
  • Genetically modified cells produced by the methods described herein may be used immediately.
  • the cells may be frozen at liquid nitrogen temperatures and stored for long periods of time, being thawed and capable of being reused.
  • the cells will usually be frozen in 10% dimethylsulfoxide (DMSO), 50% serum, 40% buffered medium, or some other such solution as is commonly used in the art to preserve cells at such freezing temperatures, and thawed in a manner as commonly known in the art for thawing frozen cultured cells.
  • DMSO dimethylsulfoxide
  • the genetically modified cells may be cultured in vitro under various culture conditions.
  • the cells may be expanded in culture, i.e. grown under conditions that promote their proliferation.
  • Culture medium may be liquid or semi-solid, e.g. containing agar, methylcellulose, etc.
  • the cell population may be suspended in an appropriate nutrient medium, such as Iscove's modified DMEM or RPMI 1640, normally supplemented with fetal calf serum (about 5-10%), L-glutamine, a thiol, particularly 2-mercaptoethanol, and antibiotics, e.g. penicillin and streptomycin.
  • the culture may contain growth factors to which the regulatory T cells are responsive.
  • Growth factors as defined herein, are molecules capable of promoting survival, growth and/or differentiation of cells, either in culture or in the intact tissue, through specific effects on a transmembrane receptor. Growth factors include polypeptides and non-polypeptide factors.
  • Cells that have been genetically modified in this way may be transplanted to a subject for purposes such as gene therapy, e.g. to treat a disease or as an antiviral, antipathogenic, or anticancer therapeutic, for the production of genetically modified organisms in agriculture, or for biological research.
  • the subject may be a neonate, a juvenile, or an adult.
  • Mammalian species that may be treated with the present methods include canines and felines; equines; bovines; ovines; etc. and primates, particularly humans.
  • Animal models, particularly small mammals e.g. mouse, rat, guinea pig, hamster, lagomorpha (e.g., rabbit), etc.
  • small mammals e.g. mouse, rat, guinea pig, hamster, lagomorpha (e.g., rabbit), etc.
  • Cells may be provided to the subject alone or with a suitable substrate or matrix, e.g. to support their growth and/or organization in the tissue to which they are being transplanted. Usually, at least 1 ⁇ 10 3 cells will be administered, for example 5 ⁇ 10 3 cells, 1 ⁇ 10 4 cells, 5 ⁇ 10 4 cells, 1 ⁇ 10 5 cells, 1 ⁇ 10 6 cells or more.
  • the cells may be introduced to the subject via any of the following routes: parenteral, subcutaneous, intravenous, intracranial, intraspinal, intraocular, or into spinal fluid.
  • the cells may be introduced by injection, catheter, or the like. Examples of methods for local delivery, that is, delivery to the site of injury, include, e.g. through an Ommaya reservoir, e.g.
  • Cells may also be introduced into an embryo (e.g., a blastocyst) for the purpose of generating a transgenic animal (e.g., a transgenic mouse).
  • the number of administrations of treatment to a subject may vary. Introducing the genetically modified cells into the subject may be a one-time event; but in certain situations, such treatment may elicit improvement for a limited period of time and require an on-going series of repeated treatments. In other situations, multiple administrations of the genetically modified cells may be required before an effect is observed.
  • the exact protocols depend upon the disease or condition, the stage of the disease and parameters of the individual subject being treated.
  • the guide RNA and/or site-directed modifying polypeptide and/or donor polynucleotide are employed to modify cellular DNA in vivo, again for purposes such as gene therapy, e.g. to treat a disease or as an antiviral, antipathogenic, or anticancer therapeutic, for the production of genetically modified organisms in agriculture, or for biological research.
  • a guide RNA and/or site-directed modifying polypeptide and/or donor polynucleotide are administered directly to the individual.
  • a guide RNA and/or site-directed modifying polypeptide and/or donor polynucleotide may be administered by any of a number of well-known methods in the art for the administration of peptides, small molecules and nucleic acids to a subject.
  • a guide RNA and/or site-directed modifying polypeptide and/or donor polynucleotide can be incorporated into a variety of formulations. More particularly, a guide RNA and/or site-directed modifying polypeptide and/or donor polynucleotide of the present invention can be formulated into pharmaceutical compositions by combination with appropriate pharmaceutically acceptable carriers or diluents.
  • compositions that include one or more a guide RNA and/or site-directed modifying polypeptide and/or donor polynucleotide present in a pharmaceutically acceptable vehicle.
  • “Pharmaceutically acceptable vehicles” may be vehicles approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, such as humans.
  • vehicle refers to a diluent, adjuvant, excipient, or carrier with which a compound of the invention is formulated for administration to a mammal.
  • Such pharmaceutical vehicles can be lipids, e.g. liposomes, e.g.
  • liposome dendrimers such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, saline; gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like.
  • auxiliary, stabilizing, thickening, lubricating and coloring agents may be used.
  • Pharmaceutical compositions may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols.
  • administration of the a guide RNA and/or site-directed modifying polypeptide and/or donor polynucleotide can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intratracheal, intraocular, etc., administration.
  • the active agent may be systemic after administration or may be localized by the use of regional administration, intramural administration, or use of an implant that acts to retain the active dose at the site of implantation.
  • the active agent may be formulated for immediate activity or it may be formulated for sustained release.
  • BBB blood-brain barrier
  • osmotic means such as mannitol or leukotrienes
  • vasoactive substances such as bradykinin.
  • a BBB disrupting agent can be co-administered with the therapeutic compositions of the invention when the compositions are administered by intravascular injection.
  • an effective amount of a guide RNA and/or site-directed modifying polypeptide and/or donor polynucleotide are provided.
  • an effective amount or effective dose of a guide RNA and/or site-directed modifying polypeptide and/or donor polynucleotide in vivo is the amount to induce a 2 fold increase or more in the amount of recombination observed between two homologous sequences relative to a negative control, e.g. a cell contacted with an empty vector or irrelevant polypeptide.
  • the amount of recombination may be measured by any convenient method, e.g. as described above and known in the art.
  • the calculation of the effective amount or effective dose of a guide RNA and/or site-directed modifying polypeptide and/or donor polynucleotide to be administered is within the skill of one of ordinary skill in the art, and will be routine to those persons skilled in the art.
  • the final amount to be administered will be dependent upon the route of administration and upon the nature of the disorder or condition that is to be treated.
  • the effective amount given to a particular patient will depend on a variety of factors, several of which will differ from patient to patient.
  • a competent clinician will be able to determine an effective amount of a therapeutic agent to administer to a patient to halt or reverse the progression the disease condition as required.
  • a clinician can determine the maximum safe dose for an individual, depending on the route of administration. For instance, an intravenously administered dose may be more than an intrathecally administered dose, given the greater body of fluid into which the therapeutic composition is being administered. Similarly, compositions which are rapidly cleared from the body may be administered at higher doses, or in repeated doses, in order to maintain a therapeutic concentration.
  • the competent clinician will be able to optimize the dosage of a particular therapeutic in the course of routine clinical trials.
  • a guide RNA and/or site-directed modifying polypeptide and/or donor polynucleotide may be obtained from a suitable commercial source.
  • the total pharmaceutically effective amount of the a guide RNA and/or site-directed modifying polypeptide and/or donor polynucleotide administered parenterally per dose will be in a range that can be measured by a dose response curve.
  • Therapies based on a guide RNA and/or site-directed modifying polypeptide and/or donor polynucleotides i.e. preparations of a guide RNA and/or site-directed modifying polypeptide and/or donor polynucleotide to be used for therapeutic administration, must be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 ⁇ m membranes).
  • Therapeutic compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • the therapies based on a guide RNA and/or site-directed modifying polypeptide and/or donor polynucleotide may be stored in unit or multi-dose containers, for example, sealed ampules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution.
  • a lyophilized formulation 10-ml vials are filled with 5 ml of sterile-filtered 1% (w/v) aqueous solution of compound, and the resulting mixture is lyophilized.
  • the infusion solution is prepared by reconstituting the lyophilized compound using bacteriostatic Water-for-Injection.
  • compositions can include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers of diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration.
  • diluents are selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, buffered water, physiological saline, PBS, Ringer's solution, dextrose solution, and Hank's solution.
  • the pharmaceutical composition or formulation can include other carriers, adjuvants, or non-toxic, nontherapeutic, nonimmunogenic stabilizers, excipients and the like.
  • the compositions can also include additional substances to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, wetting agents and detergents.
  • the composition can also include any of a variety of stabilizing agents, such as an antioxidant for example.
  • the polypeptide can be complexed with various well-known compounds that enhance the in vivo stability of the polypeptide, or otherwise enhance its pharmacological properties (e.g., increase the half-life of the polypeptide, reduce its toxicity, enhance solubility or uptake). Examples of such modifications or complexing agents include sulfate, gluconate, citrate and phosphate.
  • the nucleic acids or polypeptides of a composition can also be complexed with molecules that enhance their in vivo attributes. Such molecules include, for example, carbohydrates, polyamines, amino acids, other peptides, ions (e.g., sodium, potassium, calcium, magnesium, manganese), and lipids.
  • the pharmaceutical compositions can be administered for prophylactic and/or therapeutic treatments.
  • Toxicity and therapeutic efficacy of the active ingredient can be determined according to standard pharmaceutical procedures in cell cultures and/or experimental animals, including, for example, determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Therapies that exhibit large therapeutic indices are preferred.
  • the data obtained from cell culture and/or animal studies can be used in formulating a range of dosages for humans.
  • the dosage of the active ingredient typically lines within a range of circulating concentrations that include the ED50 with low toxicity.
  • the dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the components used to formulate the pharmaceutical compositions are preferably of high purity and are substantially free of potentially harmful contaminants (e.g., at least National Food (NF) grade, generally at least analytical grade, and more typically at least pharmaceutical grade).
  • NF National Food
  • compositions intended for in vivo use are usually sterile.
  • compositions for parental administration are also sterile, substantially isotonic and made under GMP conditions.
  • the effective amount of a therapeutic composition to be given to a particular patient will depend on a variety of factors, several of which will differ from patient to patient.
  • a competent clinician will be able to determine an effective amount of a therapeutic agent to administer to a patient to halt or reverse the progression the disease condition as required.
  • a clinician can determine the maximum safe dose for an individual, depending on the route of administration. For instance, an intravenously administered dose may be more than an intrathecally administered dose, given the greater body of fluid into which the therapeutic composition is being administered. Similarly, compositions which are rapidly cleared from the body may be administered at higher doses, or in repeated doses, in order to maintain a therapeutic concentration.
  • the competent clinician will be able to optimize the dosage of a particular therapeutic in the course of routine clinical trials.
  • the present disclosure provides genetically modified host cells, including isolated genetically modified host cells, where a genetically modified host cell comprises (has been genetically modified with: 1) an exogenous guide RNA; 2) an exogenous nucleic acid comprising a nucleotide sequence encoding a guide RNA; 3) an exogenous site-directed modifying polypeptide (e.g., a naturally occurring Cas9; a modified, i.e., mutated or variant, Cas9; a chimeric Cas9; etc.); 4) an exogenous nucleic acid comprising a nucleotide sequence encoding a site-directed modifying polypeptide; or 5) any combination of the above.
  • a genetically modified host cell comprises (has been genetically modified with: 1) an exogenous guide RNA; 2) an exogenous nucleic acid comprising a nucleotide sequence encoding a guide RNA; 3) an exogenous site-directed modifying polypeptide (e.g., a naturally occurring
  • a genetically modified cell is generated by genetically modifying a host cell with, for example: 1) an exogenous guide RNA; 2) an exogenous nucleic acid comprising a nucleotide sequence encoding a guide RNA; 3) an exogenous site-directed modifying polypeptide; 4) an exogenous nucleic acid comprising a nucleotide sequence encoding a site-directed modifying polypeptide; or 5) any combination of the above.).
  • a genetically modified host cells of interest can be a cell from any organism (e.g. a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a plant cell, an algal cell, e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C. Agardh , and the like, a fungal cell (e.g., a yeast cell), an animal cell, a cell from an invertebrate animal (e.g.
  • organism e.g. a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a plant cell, an algal cell, e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Ch
  • a cell from a vertebrate animal e.g., fish, amphibian, reptile, bird, mammal
  • a cell from a mammal e.g., a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, a non-human primate, a human, etc.
  • a vertebrate animal e.g., fish, amphibian, reptile, bird, mammal
  • a cell from a mammal e.g., a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, a non-human primate, a human, etc.
  • a genetically modified host cell has been genetically modified with an exogenous nucleic acid comprising a nucleotide sequence encoding a site-directed modifying polypeptide (e.g., a naturally occurring Cas9; a modified, i.e., mutated or variant, Cas9; a chimeric Cas9; etc.).
  • a site-directed modifying polypeptide e.g., a naturally occurring Cas9; a modified, i.e., mutated or variant, Cas9; a chimeric Cas9; etc.
  • the DNA of a genetically modified host cell can be targeted for modification by introducing into the cell a guide RNA (or a DNA encoding a guide RNA, which determines the genomic location/sequence to be modified) and optionally a donor nucleic acid.
  • the nucleotide sequence encoding a site-directed modifying polypeptide is operably linked to an inducible promoter (e.g., heat shock promoter, Tetracycline-regulated promoter, Steroid-regulated promoter, Metal-regulated promoter, estrogen receptor-regulated promoter, etc.).
  • the nucleotide sequence encoding a site-directed modifying polypeptide is operably linked to a spatially restricted and/or temporally restricted promoter (e.g., a tissue specific promoter, a cell type specific promoter, etc.).
  • the nucleotide sequence encoding a site-directed modifying polypeptide is operably linked to a constitutive promoter.
  • a genetically modified host cell is in vitro. In some embodiments, a genetically modified host cell is in vivo. In some embodiments, a genetically modified host cell is a prokaryotic cell or is derived from a prokaryotic cell. In some embodiments, a genetically modified host cell is a bacterial cell or is derived from a bacterial cell. In some embodiments, a genetically modified host cell is an archaeal cell or is derived from an archaeal cell. In some embodiments, a genetically modified host cell is a eukaryotic cell or is derived from a eukaryotic cell. In some embodiments, a genetically modified host cell is a plant cell or is derived from a plant cell.
  • a genetically modified host cell is an animal cell or is derived from an animal cell. In some embodiments, a genetically modified host cell is an invertebrate cell or is derived from an invertebrate cell. In some embodiments, a genetically modified host cell is a vertebrate cell or is derived from a vertebrate cell. In some embodiments, a genetically modified host cell is a mammalian cell or is derived from a mammalian cell. In some embodiments, a genetically modified host cell is a rodent cell or is derived from a rodent cell. In some embodiments, a genetically modified host cell is a human cell or is derived from a human cell.
  • the present disclosure further provides progeny of a genetically modified cell, where the progeny can comprise the same exogenous nucleic acid or polypeptide as the genetically modified cell from which it was derived.
  • the present disclosure further provides a composition comprising a genetically modified host cell.
  • a genetically modified host cell is a genetically modified stem cell or progenitor cell.
  • Suitable host cells include, e.g., stem cells (adult stem cells, embryonic stem cells, iPS cells, etc.) and progenitor cells (e.g., cardiac progenitor cells, neural progenitor cells, etc.).
  • Suitable host cells include mammalian stem cells and progenitor cells, including, e.g., rodent stem cells, rodent progenitor cells, human stem cells, human progenitor cells, etc.
  • Suitable host cells include in vitro host cells, e.g., isolated host cells.
  • a genetically modified host cell comprises an exogenous guide RNA nucleic acid. In some embodiments, a genetically modified host cell comprises an exogenous nucleic acid comprising a nucleotide sequence encoding a guide RNA. In some embodiments, a genetically modified host cell comprises an exogenous site-directed modifying polypeptide (e.g., a naturally occurring Cas9; a modified, i.e., mutated or variant, Cas9; a chimeric Cas9; etc.). In some embodiments, a genetically modified host cell comprises an exogenous nucleic acid comprising a nucleotide sequence encoding a site-directed modifying polypeptide. In some embodiments, a genetically modified host cell comprises exogenous nucleic acid comprising a nucleotide sequence encoding 1) a guide RNA and 2) a site-directed modifying polypeptide.
  • site-directed modifying polypeptide e.g., a naturally occurring Cas9; a
  • the site-directed modifying polypeptide comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%, amino acid sequence identity to amino acids 7-166 and/or 731-1003 of SEQ ID NO: 8, or to the corresponding portions in any of the amino acid sequences set forth as SEQ ID NOs: 1-800.
  • the present disclosure provides a composition comprising a guide RNA and/or a site-directed modifying polypeptide.
  • the site-directed modifying polypeptide is a chimeric polypeptide.
  • a composition is useful for carrying out a method of the present disclosure, e.g., a method for site-specific modification of a target DNA; a method for site-specific modification of a polypeptide associated with a target DNA; etc.
  • compositions Comprising a Guide RNA
  • the present disclosure provides a composition comprising a guide RNA.
  • the composition can comprise, in addition to the guide RNA, one or more of: a salt, e.g., NaCl, MgCl 2 , KCl, MgSO 4 , etc.; a buffering agent, e.g., a Tris buffer, N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES), MES sodium salt, 3-(N-Morpholino)propanesulfonic acid (MOPS), N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; a solubilizing agent; a detergent, e.g., a non-ionic detergent such as Tween-20, etc.; a nuclease inhibitor; and the like.
  • a guide RNA present in a composition is pure, e.g., at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more than 99% pure, where “% purity” means that guide RNA is the recited percent free from other macromolecules, or contaminants that may be present during the production of the guide RNA.
  • compositions Comprising a Chimeric Polypeptide
  • the present disclosure provides a composition a chimeric polypeptide.
  • the composition can comprise, in addition to the guide RNA, one or more of: a salt, e.g., NaCl, MgCl 2 , KCl, MgSO 4 , etc.; a buffering agent, e.g., a Tris buffer, HEPES, MES, MES sodium salt, MOPS, TAPS, etc.; a solubilizing agent; a detergent, e.g., a non-ionic detergent such as Tween-20, etc.; a protease inhibitor; a reducing agent (e.g., dithiothreitol); and the like.
  • a salt e.g., NaCl, MgCl 2 , KCl, MgSO 4 , etc.
  • a buffering agent e.g., a Tris buffer, HEPES, MES, MES sodium salt, MOPS, TAPS, etc.
  • a chimeric polypeptide present in a composition is pure, e.g., at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more than 99% pure, where “% purity” means that the site-directed modifying polypeptide is the recited percent free from other proteins, other macromolecules, or contaminants that may be present during the production of the chimeric polypeptide.
  • compositions Comprising a Guide RNA and a Site-Directed Modifying Polypeptide
  • the present disclosure provides a composition comprising: (i) a guide RNA or a DNA polynucleotide encoding the same; and ii) a site-directed modifying polypeptide, or a polynucleotide encoding the same.
  • the site-directed modifying polypeptide is a chimeric site-directed modifying polypeptide.
  • the site-directed modifying polypeptide is a naturally-occurring site-directed modifying polypeptide.
  • the site-directed modifying polypeptide exhibits enzymatic activity that modifies a target DNA.
  • the site-directed modifying polypeptide exhibits enzymatic activity that modifies a polypeptide that is associated with a target DNA.
  • the site-directed modifying polypeptide modulates transcription of the target DNA.
  • the present disclosure provides a composition
  • a composition comprising: (i) a guide RNA, as described above, or a DNA polynucleotide encoding the same, the guide RNA comprising: (a) a first segment comprising a nucleotide sequence that is complementary to a sequence in a target DNA; and (b) a second segment that interacts with a site-directed modifying polypeptide; and (ii) the site-directed modifying polypeptide, or a polynucleotide encoding the same, the site-directed modifying polypeptide comprising: (a) an RNA-binding portion that interacts with the guide RNA; and (b) an activity portion that exhibits site-directed enzymatic activity, wherein the site of enzymatic activity is determined by the guide RNA.
  • a composition comprises: a composition comprising: (i) a guide RNA, the guide RNA comprising: (a) a first segment comprising a nucleotide sequence that is complementary to a sequence in a target DNA; and (b) a second segment that interacts with a site-directed modifying polypeptide; and (ii) the site-directed modifying polypeptide, the site-directed modifying polypeptide comprising: (a) an RNA-binding portion that interacts with the guide RNA; and (b) an activity portion that exhibits site-directed enzymatic activity, wherein the site of enzymatic activity is determined by the guide RNA.
  • a composition comprises: (i) a polynucleotide encoding a guide RNA, the guide RNA comprising: (a) a first segment comprising a nucleotide sequence that is complementary to a sequence in a target DNA; and (b) a second segment that interacts with a site-directed modifying polypeptide; and (ii) a polynucleotide encoding the site-directed modifying polypeptide, the site-directed modifying polypeptide comprising: (a) an RNA-binding portion that interacts with the guide RNA; and (b) an activity portion that exhibits site-directed enzymatic activity, wherein the site of enzymatic activity is determined by the guide RNA.
  • a composition includes both RNA molecules of a double-molecule guide RNA.
  • a composition includes an activator-RNA that comprises a duplex-forming segment that is complementary to the duplex-forming segment of a targeter-.
  • the duplex-forming segments of the activator-RNA and the targeter-RNA hybridize to form the dsRNA duplex of the protein-binding segment of the guide RNA.
  • the targeter-RNA further provides the DNA-targeting segment (single stranded) of the guide RNA and therefore targets the guide RNA to a specific sequence within the target DNA.
  • the duplex-forming segment of the activator-RNA comprises a nucleotide sequence that has at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or 100% identity with a tracrRNA sequence set out in Supplementary Table S5.
  • the duplex-forming segment of the targeter-RNA comprises a nucleotide sequence that has at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or 100% identity with a CRISPR repeat sequence set out in Supplementary Table S5.
  • the present disclosure provides a composition comprising: (i) a guide RNA, or a DNA polynucleotide encoding the same, the guide RNA comprising: (a) a first segment comprising a nucleotide sequence that is complementary to a sequence in a target DNA; and (b) a second segment that interacts with a site-directed modifying polypeptide; and (ii) the site-directed modifying polypeptide, or a polynucleotide encoding the same, the site-directed modifying polypeptide comprising: (a) an RNA-binding portion that interacts with the guide RNA; and (b) an activity portion that modulates transcription within the target DNA, wherein the site of modulated transcription within the target DNA is determined by the guide RNA.
  • a composition comprises: (i) a guide RNA, the guide RNA comprising: (a) a first segment comprising a nucleotide sequence that is complementary to a sequence in a target DNA; and (b) a second segment that interacts with a site-directed modifying polypeptide; and (ii) the site-directed modifying polypeptide, the site-directed modifying polypeptide comprising: (a) an RNA-binding portion that interacts with the guide RNA; and (b) an activity portion that modulates transcription within the target DNA, wherein the site of modulated transcription within the target DNA is determined by the guide RNA.
  • a composition comprises: (i) a DNA polynucleotide encoding a guide RNA, the guide RNA comprising: (a) a first segment comprising a nucleotide sequence that is complementary to a sequence in a target DNA; and (b) a second segment that interacts with a site-directed modifying polypeptide; and (ii) a polynucleotide encoding the site-directed modifying polypeptide, the site-directed modifying polypeptide comprising: (a) an RNA-binding portion that interacts with the guide RNA; and (b) an activity portion that modulates transcription within the target DNA, wherein the site of modulated transcription within the target DNA is determined by the guide RNA.
  • a composition can comprise, in addition to i) a guide RNA, or a DNA polynucleotide encoding the same; and ii) a site-directed modifying polypeptide, or a polynucleotide encoding the same, one or more of: a salt, e.g., NaCl, MgCl 2 , KCl, MgSO 4 , etc.; a buffering agent, e.g., a Tris buffer, HEPES, MES, MES sodium salt, MOPS, TAPS, etc.; a solubilizing agent; a detergent, e.g., a non-ionic detergent such as Tween-20, etc.; a protease inhibitor; a reducing agent (e.g., dithiothreitol); and the like.
  • a salt e.g., NaCl, MgCl 2 , KCl, MgSO 4 , etc.
  • a buffering agent e
  • the components of the composition are individually pure, e.g., each of the components is at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least 99%, pure. In some cases, the individual components of a composition are pure before being added to the composition.
  • a site-directed modifying polypeptide present in a composition is pure, e.g., at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more than 99% pure, where “A, purity” means that the site-directed modifying polypeptide is the recited percent free from other proteins (e.g., proteins other than the site-directed modifying polypeptide), other macromolecules, or contaminants that may be present during the production of the site-directed modifying polypeptide.
  • kits for carrying out a method can include one or more of: a site-directed modifying polypeptide; a nucleic acid comprising a nucleotide encoding a site-directed modifying polypeptide; a guide RNA; a nucleic acid comprising a nucleotide sequence encoding a guide RNA; an activator-RNA; a nucleic acid comprising a nucleotide sequence encoding an activator-RNA; a targeter-RNA; and a nucleic acid comprising a nucleotide sequence encoding a targeter-RNA.
  • a site-directed modifying polypeptide; a nucleic acid comprising a nucleotide encoding a site-directed modifying polypeptide; a guide RNA; a nucleic acid comprising a nucleotide sequence encoding a guide RNA; an activator-RNA; a nucleic acid comprising a nucleotide sequence encoding an activator-RNA; a targeter-RNA; and a nucleic acid comprising a nucleotide sequence encoding a targeter-RNA, are described in detail above.
  • a kit may comprise a complex that comprises two or more of: a site-directed modifying polypeptide; a nucleic acid comprising a nucleotide encoding a site-directed modifying polypeptide; a guide RNA; a nucleic acid comprising a nucleotide sequence encoding a guide RNA; an activator-RNA; a nucleic acid comprising a nucleotide sequence encoding an activator-RNA; a targeter-RNA; and a nucleic acid comprising a nucleotide sequence encoding a targeter-RNA.
  • a kit comprises a site-directed modifying polypeptide, or a polynucleotide encoding the same.
  • the site-directed modifying polypeptide comprises: (a) an RNA-binding portion that interacts with the guide RNA; and (b) an activity portion that modulates transcription within the target DNA, wherein the site of modulated transcription within the target DNA is determined by the guide RNA.
  • the activity portion of the site-directed modifying polypeptide exhibits reduced or inactivated nuclease activity.
  • the site-directed modifying polypeptide is a chimeric site-directed modifying polypeptide.
  • a kit comprises: a site-directed modifying polypeptide, or a polynucleotide encoding the same, and a reagent for reconstituting and/or diluting the site-directed modifying polypeptide.
  • a kit comprises a nucleic acid (e.g., DNA, RNA) comprising a nucleotide encoding a site-directed modifying polypeptide.
  • a kit comprises: a nucleic acid (e.g., DNA, RNA) comprising a nucleotide encoding a site-directed modifying polypeptide; and a reagent for reconstituting and/or diluting the site-directed modifying polypeptide.
  • a kit comprising a site-directed modifying polypeptide, or a polynucleotide encoding the same can further include one or more additional reagents, where such additional reagents can be selected from: a buffer for introducing the site-directed modifying polypeptide into a cell; a wash buffer; a control reagent; a control expression vector or RNA polynucleotide; a reagent for in vitro production of the site-directed modifying polypeptide from DNA, and the like.
  • the site-directed modifying polypeptide included in a kit is a chimeric site-directed modifying polypeptide, as described above.
  • a kit comprises a guide RNA, or a DNA polynucleotide encoding the same, the guide RNA comprising: (a) a first segment comprising a nucleotide sequence that is complementary to a sequence in a target DNA; and (b) a second segment that interacts with a site-directed modifying polypeptide.
  • the guide RNA further comprises a third segment (as described above).
  • a kit comprises: (i) a guide RNA, or a DNA polynucleotide encoding the same, the guide RNA comprising: (a) a first segment comprising a nucleotide sequence that is complementary to a sequence in a target DNA; and (b) a second segment that interacts with a site-directed modifying polypeptide; and (ii) a site-directed modifying polypeptide, or a polynucleotide encoding the same, the site-directed modifying polypeptide comprising: (a) an RNA-binding portion that interacts with the guide RNA; and (b) an activity portion that exhibits site-directed enzymatic activity, wherein the site of enzymatic activity is determined by the guide RNA.
  • the activity portion of the site-directed modifying polypeptide does not exhibit enzymatic activity (comprises an inactivated nuclease, e.g., via mutation).
  • the kit comprises a guide RNA and a site-directed modifying polypeptide.
  • the kit comprises: (i) a nucleic acid comprising a nucleotide sequence encoding a guide RNA; and (ii) a nucleic acid comprising a nucleotide sequence encoding site-directed modifying polypeptide.
  • a kit can include: (i) a guide RNA, or a DNA polynucleotide encoding the same, comprising: (a) a first segment comprising a nucleotide sequence that is complementary to a sequence in a target DNA; and (b) a second segment that interacts with a site-directed modifying polypeptide; and (ii) the site-directed modifying polypeptide, or a polynucleotide encoding the same, comprising: (a) an RNA-binding portion that interacts with the guide RNA; and (b) an activity portion that that modulates transcription within the target DNA, wherein the site of modulated transcription within the target DNA is determined by the guide RNA
  • the kit comprises: (i) a guide RNA; and a site-directed modifying polypeptide.
  • the kit comprises: (i) a nucleic acid comprising a nucleotide sequence encoding a guide RNA; and (ii) a nucleic acid comprising a nucleotide sequence encoding site-directed modifying polypeptide.
  • the present disclosure provides a kit comprising: (1) a recombinant expression vector comprising (i) a nucleotide sequence encoding a guide RNA, wherein the guide RNA comprises: (a) a first segment comprising a nucleotide sequence that is complementary to a sequence in a target DNA; and (b) a second segment that interacts with a site-directed modifying polypeptide; and (ii) a nucleotide sequence encoding the site-directed modifying polypeptide, wherein the site-directed modifying polypeptide comprises: (a) an RNA-binding portion that interacts with the guide RNA; and (b) an activity portion that exhibits site-directed enzymatic activity, wherein the site of enzymatic activity is determined by the guide RNA; and (2) a reagent for reconstitution and/or dilution of the expression vector.
  • the present disclosure provides a kit comprising: (1) a recombinant expression vector comprising: (i) a nucleotide sequence encoding a guide RNA, wherein the guide RNA comprises: (a) a first segment comprising a nucleotide sequence that is complementary to a sequence in a target DNA; and (b) a second segment that interacts with a site-directed modifying polypeptide; and (ii) a nucleotide sequence encoding the site-directed modifying polypeptide, wherein the site-directed modifying polypeptide comprises: (a) an RNA-binding portion that interacts with the guide RNA; and (b) an activity portion that modulates transcription within the target DNA, wherein the site of modulated transcription within the target DNA is determined by the guide RNA; and (2) a reagent for reconstitution and/or dilution of the recombinant expression vector.
  • the present disclosure provides a kit comprising: (1) a recombinant expression vector comprising a nucleic acid comprising a nucleotide sequence that encodes a DNA targeting RNA comprising: (i) a first segment comprising a nucleotide sequence that is complementary to a sequence in a target DNA; and (ii) a second segment that interacts with a site-directed modifying polypeptide; and (2) a reagent for reconstitution and/or dilution of the recombinant expression vector.
  • the kit comprises: a recombinant expression vector comprising a nucleotide sequence that encodes a site-directed modifying polypeptide, wherein the site-directed modifying polypeptide comprises: (a) an RNA-binding portion that interacts with the guide RNA; and (b) an activity portion that exhibits site-directed enzymatic activity, wherein the site of enzymatic activity is determined by the guide RNA.
  • the kit comprises: a recombinant expression vector comprising a nucleotide sequence that encodes a site-directed modifying polypeptide, wherein the site-directed modifying polypeptide comprises: (a) an RNA-binding portion that interacts with the guide RNA; and (b) an activity portion that modulates transcription within the target DNA, wherein the site of modulated transcription within the target DNA is determined by the guide RNA.
  • the kit comprises an activator-RNA or a targeter-RNA. In some embodiments of any of the above kits, the kit comprises a single-molecule guide RNA. In some embodiments of any of the above kits, the kit comprises two or more double-molecule or single-molecule guide RNAs. In some embodiments of any of the above kits, a guide RNA (e.g., including two or more guide RNAs) can be provided as an array (e.g., an array of RNA molecules, an array of DNA molecules encoding the guide RNA(s), etc.). Such kits can be useful, for example, for use in conjunction with the above described genetically modified host cells that comprise a site-directed modifying polypeptide. In some embodiments of any of the above kits, the kit further comprises a donor polynucleotide to effect the desired genetic modification. Components of a kit can be in separate containers; or can be combined in a single container.
  • kits further comprises one or more variant Cas9 site-directed polypeptides that exhibits reduced endodeoxyribonuclease activity relative to wild-type Cas9.
  • kits further comprises one or more nucleic acids comprising a nucleotide sequence encoding a variant Cas9 site-directed polypeptide that exhibits reduced endodeoxyribonuclease activity relative to wild-type Cas9.
  • kits can further include one or more additional reagents, where such additional reagents can be selected from: a dilution buffer; a reconstitution solution; a wash buffer; a control reagent; a control expression vector or RNA polynucleotide; a reagent for in vitro production of the site-directed modifying polypeptide from DNA, and the like.
  • additional reagents can be selected from: a dilution buffer; a reconstitution solution; a wash buffer; a control reagent; a control expression vector or RNA polynucleotide; a reagent for in vitro production of the site-directed modifying polypeptide from DNA, and the like.
  • a kit can further include instructions for using the components of the kit to practice the methods.
  • the instructions for practicing the methods are generally recorded on a suitable recording medium.
  • the instructions may be printed on a substrate, such as paper or plastic, etc.
  • the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging) etc.
  • the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, flash drive, etc.
  • the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the Internet, are provided.
  • An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.
  • a genetically modified host cell has been genetically modified with an exogenous nucleic acid comprising a nucleotide sequence encoding a site-directed modifying polypeptide (e.g., a naturally occurring Cas9; a modified, i.e., mutated or variant, Cas9; a chimeric Cas9; etc.). If such a cell is a eukaryotic single-cell organism, then the modified cell can be considered a genetically modified organism.
  • the non-human genetically modified organism is a Cas9 transgenic multicellular organism.
  • a genetically modified non-human host cell e.g., a cell that has been genetically modified with an exogenous nucleic acid comprising a nucleotide sequence encoding a site-directed modifying polypeptide, e.g., a naturally occurring Cas9; a modified, i.e., mutated or variant, Cas9; a chimeric Cas9; etc.
  • a genetically modified nonhuman organism e.g., a mouse, a fish, a frog, a fly, a worm, etc.
  • the genetically modified host cell is a pluripotent stem cell (i.e., PSC) or a germ cell (e.g., sperm, oocyte, etc.)
  • a pluripotent stem cell i.e., PSC
  • a germ cell e.g., sperm, oocyte, etc.
  • an entire genetically modified organism can be derived from the genetically modified host cell.
  • the genetically modified host cell is a pluripotent stem cell (e.g., ESC, iPSC, pluripotent plant stem cell, etc.) or a germ cell (e.g., sperm cell, oocyte, etc.), either in vivo or in vitro, that can give rise to a genetically modified organism.
  • the genetically modified host cell is a vertebrate PSC (e.g., ESC, iPSC, etc.) and is used to generate a genetically modified organism (e.g. by injecting a PSC into a blastocyst to produce a chimeric/mosaic animal, which could then be mated to generate non-chimeric/non-mosaic genetically modified organisms; grafting in the case of plants; etc.).
  • a vertebrate PSC e.g., ESC, iPSC, etc.
  • a genetically modified organism e.g. by injecting a PSC into a blastocyst to produce a chimeric/mosaic animal, which could then be mated to generate non-chimeric/non-mosaic genetically modified organisms; grafting in the case of plants; etc.
  • Any convenient method/protocol for producing a genetically modified organism is suitable for producing a genetically modified host cell comprising an exogenous nucleic acid comprising a nucleotide sequence encoding a site-directed modifying polypeptide (e.g., a naturally occurring Cas9; a modified, i.e., mutated or variant, Cas9; a chimeric Cas9; etc.).
  • a site-directed modifying polypeptide e.g., a naturally occurring Cas9; a modified, i.e., mutated or variant, Cas9; a chimeric Cas9; etc.
  • Methods of producing genetically modified organisms are known in the art. For example, see Cho et al., Curr Protoc Cell Biol. 2009 March; Chapter 19:Unit 19.11: Generation of transgenic mice; Gama et al., Brain Struct Funct.
  • a genetically modified organism comprises a target cell for methods of the invention, and thus can be considered a source for target cells.
  • a genetically modified cell comprising an exogenous nucleic acid comprising a nucleotide sequence encoding a site-directed modifying polypeptide (e.g., a naturally occurring Cas9; a modified, i.e., mutated or variant, Cas9; a chimeric Cas9; etc.) is used to generate a genetically modified organism, then the cells of the genetically modified organism comprise the exogenous nucleic acid comprising a nucleotide sequence encoding a site-directed modifying polypeptide (e.g., a naturally occurring Cas9; a modified, i.e., mutated or variant, Cas9; a chimeric Cas9; etc.).
  • a site-directed modifying polypeptide e.g., a naturally occurring Cas9; a modified, i.e., mutated or
  • the DNA of a cell or cells of the genetically modified organism can be targeted for modification by introducing into the cell or cells a guide RNA (or a DNA encoding a guide RNA) and optionally a donor nucleic acid.
  • a guide RNA or a DNA encoding a guide RNA
  • the introduction of a guide RNA (or a DNA encoding a guide RNA) into a subset of cells (e.g., brain cells, intestinal cells, kidney cells, lung cells, blood cells, etc.) of the genetically modified organism can target the DNA of such cells for modification, the genomic location of which will depend on the DNA-targeting sequence of the introduced guide RNA.
  • a genetically modified organism is a source of target cells for methods of the invention.
  • a genetically modified organism comprising cells that are genetically modified with an exogenous nucleic acid comprising a nucleotide sequence encoding a site-directed modifying polypeptide (e.g., a naturally occurring Cas9; a modified, i.e., mutated or variant, Cas9; a chimeric Cas9; etc.) can provide a source of genetically modified cells, for example PSCs (e.g., ESCs, iPSCs, sperm, oocytes, etc.), neurons, progenitor cells, cardiomyocytes, etc.
  • PSCs e.g., ESCs, iPSCs, sperm, oocytes, etc.
  • a genetically modified cell is a PSC comprising an exogenous nucleic acid comprising a nucleotide sequence encoding a site-directed modifying polypeptide (e.g., a naturally occurring Cas9; a modified, i.e., mutated or variant, Cas9; a chimeric Cas9; etc.).
  • a site-directed modifying polypeptide e.g., a naturally occurring Cas9; a modified, i.e., mutated or variant, Cas9; a chimeric Cas9; etc.
  • the PSC can be a target cell such that the DNA of the PSC can be targeted for modification by introducing into the PSC a guide RNA (or a DNA encoding a guide RNA) and optionally a donor nucleic acid, and the genomic location of the modification will depend on the DNA-targeting sequence of the introduced guide RNA.
  • the methods described herein can be used to modify the DNA (e.g., delete and/or replace any desired genomic location) of PSCs derived from a genetically modified organism.
  • modified PSCs can then be used to generate organisms having both (i) an exogenous nucleic acid comprising a nucleotide sequence encoding a site-directed modifying polypeptide (e.g., a naturally occurring Cas9; a modified, i.e., mutated or variant, Cas9; a chimeric Cas9; etc.) and (ii) a DNA modification that was introduced into the PSC.
  • a site-directed modifying polypeptide e.g., a naturally occurring Cas9; a modified, i.e., mutated or variant, Cas9; a chimeric Cas9; etc.
  • An exogenous nucleic acid comprising a nucleotide sequence encoding a site-directed modifying polypeptide (e.g., a naturally occurring Cas9; a modified, i.e., mutated or variant, Cas9; a chimeric Cas9; etc.) can be under the control of (i.e., operably linked to) an unknown promoter (e.g., when the nucleic acid randomly integrates into a host cell genome) or can be under the control of (i.e., operably linked to) a known promoter.
  • a site-directed modifying polypeptide e.g., a naturally occurring Cas9; a modified, i.e., mutated or variant, Cas9; a chimeric Cas9; etc.
  • an unknown promoter e.g., when the nucleic acid randomly integrates into a host cell genome
  • a known promoter e.g., when the nucleic acid randomly integrates into a host
  • Suitable known promoters can be any known promoter and include constitutively active promoters (e.g., CMV promoter), inducible promoters (e.g., heat shock promoter, Tetracycline-regulated promoter, Steroid-regulated promoter, Metal-regulated promoter, estrogen receptor-regulated promoter, etc.), spatially restricted and/or temporally restricted promoters (e.g., a tissue specific promoter, a cell type specific promoter, etc.), etc.
  • constitutively active promoters e.g., CMV promoter
  • inducible promoters e.g., heat shock promoter, Tetracycline-regulated promoter, Steroid-regulated promoter, Metal-regulated promoter, estrogen receptor-regulated promoter, etc.
  • spatially restricted and/or temporally restricted promoters e.g., a tissue specific promoter, a cell type specific promoter, etc.
  • a genetically modified organism e.g. an organism whose cells comprise a nucleotide sequence encoding a site-directed modifying polypeptide, e.g., a naturally occurring Cas9; a modified, i.e., mutated or variant, Cas9; a chimeric Cas9; etc.
  • a plant e.g., a plant; algae; an invertebrate (e.g., a cnidarian, an echinoderm, a worm, a fly, etc.); a vertebrate (e.g., a fish (e.g., zebrafish, puffer fish, gold fish, etc.), an amphibian (e.g., salamander, frog, etc.), a reptile, a bird, a mammal, etc.); an ungulate (e.g., a goat, a pig, a sheep, a cow, etc.); a rodent (e.g.,
  • the site-directed modifying polypeptide comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%, amino acid sequence identity to amino acids 7-166 and/or 731-1003 of SEQ ID NO: 8, or to the corresponding portions in any of the amino acid sequences set forth as SEQ ID NOs: 1-800.
  • a nucleic acid e.g., a nucleotide sequence encoding a site-directed modifying polypeptide, e.g., a naturally occurring Cas9; a modified, i.e., mutated or variant, Cas9; a chimeric Cas9; etc.
  • a recombinant expression vector is used as a transgene to generate a transgenic animal that produces a site-directed modifying polypeptide.
  • the present disclosure further provides a transgenic non-human animal, which animal comprises a transgene comprising a nucleic acid comprising a nucleotide sequence encoding a site-directed modifying polypeptide, e.g., a naturally occurring Cas9; a modified, i.e., mutated or variant, Cas9; a chimeric Cas9; etc., as described above.
  • the genome of the transgenic non-human animal comprises a nucleotide sequence encoding a site-directed modifying polypeptide.
  • the transgenic non-human animal is homozygous for the genetic modification. In some embodiments, the transgenic non-human animal is heterozygous for the genetic modification.
  • the transgenic non-human animal is a vertebrate, for example, a fish (e.g., zebra fish, gold fish, puffer fish, cave fish, etc.), an amphibian (frog, salamander, etc.), a bird (e.g., chicken, turkey, etc.), a reptile (e.g., snake, lizard, etc.), a mammal (e.g., an ungulate, e.g., a pig, a cow, a goat, a sheep, etc.; a lagomorph (e.g., a rabbit); a rodent (e.g., a rat, a mouse); a nonhuman primate; etc.), etc.
  • a fish e.g., zebra fish, gold fish, puffer fish, cave fish, etc.
  • an amphibian frog, salamander, etc.
  • a bird e.g., chicken, turkey, etc.
  • a reptile e.g.
  • An exogenous nucleic acid comprising a nucleotide sequence encoding a site-directed modifying polypeptide (e.g., a naturally occurring Cas9; a modified, i.e., mutated or variant, Cas9; a chimeric Cas9; etc.) can be under the control of (i.e., operably linked to) an unknown promoter (e.g., when the nucleic acid randomly integrates into a host cell genome) or can be under the control of (i.e., operably linked to) a known promoter.
  • a site-directed modifying polypeptide e.g., a naturally occurring Cas9; a modified, i.e., mutated or variant, Cas9; a chimeric Cas9; etc.
  • an unknown promoter e.g., when the nucleic acid randomly integrates into a host cell genome
  • a known promoter e.g., when the nucleic acid randomly integrates into a host
  • Suitable known promoters can be any known promoter and include constitutively active promoters (e.g., CMV promoter), inducible promoters (e.g., heat shock promoter, Tetracycline-regulated promoter, Steroid-regulated promoter, Metal-regulated promoter, estrogen receptor-regulated promoter, etc.), spatially restricted and/or temporally restricted promoters (e.g., a tissue specific promoter, a cell type specific promoter, etc.), etc.
  • constitutively active promoters e.g., CMV promoter
  • inducible promoters e.g., heat shock promoter, Tetracycline-regulated promoter, Steroid-regulated promoter, Metal-regulated promoter, estrogen receptor-regulated promoter, etc.
  • spatially restricted and/or temporally restricted promoters e.g., a tissue specific promoter, a cell type specific promoter, etc.
  • the site-directed modifying polypeptide comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%, amino acid sequence identity to amino acids 7-166 and/or 731-1003 of SEQ ID NO: 8, or to the corresponding portions in any of the amino acid sequences set forth as SEQ ID NOs: 1-800.
  • a nucleic acid e.g., a nucleotide sequence encoding a site-directed modifying polypeptide, e.g., a naturally occurring Cas9; a modified, i.e., mutated or variant, Cas9; a chimeric Cas9; etc.
  • a recombinant expression vector is used as a transgene to generate a transgenic plant that produces a site-directed modifying polypeptide.
  • the present disclosure further provides a transgenic plant, which plant comprises a transgene comprising a nucleic acid comprising a nucleotide sequence encoding site-directed modifying polypeptide, e.g., a naturally occurring Cas9; a modified, i.e., mutated or variant, Cas9; a chimeric Cas9; etc., as described above.
  • site-directed modifying polypeptide e.g., a naturally occurring Cas9; a modified, i.e., mutated or variant, Cas9; a chimeric Cas9; etc.
  • the genome of the transgenic plant comprises a nucleic acid.
  • the transgenic plant is homozygous for the genetic modification.
  • the transgenic plant is heterozygous for the genetic modification.
  • Suitable methods include viral infection (such as double stranded DNA viruses), transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate precipitation, direct microinjection, silicon carbide whiskers technology, Agrobacterium -mediated transformation and the like.
  • the choice of method is generally dependent on the type of cell being transformed and the circumstances under which the transformation is taking place (i.e. in vitro, ex vivo, or in vivo). Transformation methods based upon the soil bacterium Agrobacterium tumefaciens are particularly useful for introducing an exogenous nucleic acid molecule into a vascular plant.
  • the wild type form of Agrobacterium contains a Ti (tumor-inducing) plasmid that directs production of tumorigenic crown gall growth on host plants. Transfer of the tumor-inducing T-DNA region of the Ti plasmid to a plant genome requires the Ti plasmid-encoded virulence genes as well as T-DNA borders, which are a set of direct DNA repeats that delineate the region to be transferred.
  • An Agrobacterium -based vector is a modified form of a Ti plasmid, in which the tumor inducing functions are replaced by the nucleic acid sequence of interest to be introduced into the plant host.
  • Agrobacterium -mediated transformation generally employs cointegrate vectors or binary vector systems, in which the components of the Ti plasmid are divided between a helper vector, which resides permanently in the Agrobacterium host and carries the virulence genes, and a shuttle vector, which contains the gene of interest bounded by T-DNA sequences.
  • binary vectors are well known in the art and are commercially available, for example, from Clontech (Palo Alto, Calif.).
  • Methods of coculturing Agrobacterium with cultured plant cells or wounded tissue such as leaf tissue, root explants, hypocotyledons, stem pieces or tubers, for example, also are well known in the art. See., e.g., Glick and Thompson, (eds.), Methods in Plant Molecular Biology and Biotechnology , Boca Raton, Fla.: CRC Press (1993).
  • Microprojectile-mediated transformation also can be used to produce a transgenic plant.
  • This method first described by Klein et al. ( Nature 327:70-73 (1987)), relies on microprojectiles such as gold or tungsten that are coated with the desired nucleic acid molecule by precipitation with calcium chloride, spermidine or polyethylene glycol.
  • the microprojectile particles are accelerated at high speed into an angiosperm tissue using a device such as the BIOLISTIC PD-1000 (Biorad; Hercules Calif.).
  • a nucleic acid may be introduced into a plant in a manner such that the nucleic acid is able to enter a plant cell(s), e.g., via an in vivo or ex vivo protocol.
  • in vivo it is meant in the nucleic acid is administered to a living body of a plant e.g. infiltration.
  • ex vivo it is meant that cells or explants are modified outside of the plant, and then such cells or organs are regenerated to a plant.
  • non-Ti vectors can be used to transfer the DNA into plants and cells by using free DNA delivery techniques.
  • transgenic plants such as wheat, rice (Christou (1991) Bio/Technology 9:957-9 and 4462) and corn (Gordon-Kamm (1990) Plant Cell 2: 603-618) can be produced.
  • An immature embryo can also be a good target tissue for monocots for direct DNA delivery techniques by using the particle gun (Weeks et al. (1993) Plant Physiol 102: 1077-1084; Vasil (1993) Bio/Technolo 10: 667-674; Wan and Lemeaux (1994) Plant Physiol 104: 37-48 and for Agrobacterium -mediated DNA transfer (Ishida et al.
  • Exemplary methods for introduction of DNA into chloroplasts are biolistic bombardment, polyethylene glycol transformation of protoplasts, and microinjection (Daniell et al Nat. Biotechnol 16:345-348, 1998; Staub et al Nat. Biotechnol 18: 333-338, 2000; O'Neill et al Plant J. 3:729-738, 1993; Knoblauch et al Nat. Biotechnol 17: 906-909; U.S. Pat. Nos. 5,451,513, 5,545,817, 5,545,818, and 5,576,198; in Intl. Application No.
  • Any vector suitable for the methods of biolistic bombardment, polyethylene glycol transformation of protoplasts and microinjection will be suitable as a targeting vector for chloroplast transformation.
  • Any double stranded DNA vector may be used as a transformation vector, especially when the method of introduction does not utilize Agrobacterium.
  • Plants which can be genetically modified include grains, forage crops, fruits, vegetables, oil seed crops, palms, forestry, and vines. Specific examples of plants which can be modified follow: maize, banana, peanut, field peas, sunflower, tomato, canola, tobacco, wheat, barley, oats, potato, soybeans, cotton, carnations, sorghum, lupin and rice.
  • transformed plant cells, tissues, plants and products that contain the transformed plant cells.
  • a feature of the transformed cells, and tissues and products that include the same is the presence of a nucleic acid integrated into the genome, and production by plant cells of a site-directed modifying polypeptide, e.g., a naturally occurring Cas9; a modified, i.e., mutated or variant, Cas9; a chimeric Cas9; etc.
  • Recombinant plant cells of the present invention are useful as populations of recombinant cells, or as a tissue, seed, whole plant, stem, fruit, leaf, root, flower, stem, tuber, grain, animal feed, a field of plants, and the like.
  • a nucleic acid comprising a nucleotide sequence encoding a site-directed modifying polypeptide can be under the control of (i.e., operably linked to) an unknown promoter (e.g., when the nucleic acid randomly integrates into a host cell genome) or can be under the control of (i.e., operably linked to) a known promoter.
  • Suitable known promoters can be any known promoter and include constitutively active promoters, inducible promoters, spatially restricted and/or temporally restricted promoters, etc.
  • the site-directed modifying polypeptide comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%, amino acid sequence identity to amino acids 7-166 and/or 731-1003 of SEQ ID NO: 8, or to the corresponding portions in any of the amino acid sequences set forth as SEQ ID NOs: 1-800.
  • reproductive material of a transgenic plant where reproductive material includes seeds, progeny plants and clonal material.
  • the present disclosure provides methods of modulating transcription of a target nucleic acid in a host cell.
  • the methods generally involve contacting the target nucleic acid with an enzymatically inactive Cas9 polypeptide and a single-guide RNA.
  • the methods are useful in a variety of applications, which are also provided.
  • a transcriptional modulation method of the present disclosure overcomes some of the drawbacks of methods involving RNAi.
  • a transcriptional modulation method of the present disclosure finds use in a wide variety of applications, including research applications, drug discovery (e.g., high throughput screening), target validation, industrial applications (e.g., crop engineering; microbial engineering, etc.), diagnostic applications, therapeutic applications, and imaging techniques.
  • the present disclosure provides a method of selectively modulating transcription of a target DNA in a host cell.
  • the method generally involves: a) introducing into the host cell: i) a guide RNA, or a nucleic acid comprising a nucleotide sequence encoding the guide RNA; and ii) a variant Cas9 site-directed polypeptide (“variant Cas9 polypeptide”), or a nucleic acid comprising a nucleotide sequence encoding the variant Cas9 polypeptide, where the variant Cas9 polypeptide exhibits reduced endodeoxyribonuclease activity.
  • variant Cas9 polypeptide variant Cas9 site-directed polypeptide
  • the guide RNA (also referred to herein as “guide RNA”; or “gRNA”) comprises: i) a first segment comprising a nucleotide sequence that is complementary to a target sequence in a target DNA; ii) a second segment that interacts with a site-directed polypeptide; and iii) a transcriptional terminator.
  • the first segment comprising a nucleotide sequence that is complementary to a target sequence in a target DNA, is referred to herein as a “targeting segment”.
  • the second segment which interacts with a site-directed polypeptide, is also referred to herein as a “protein-binding sequence” or “dCas9-binding hairpin,” or “dCas9 handle.”
  • segment it is meant a segment/section/region of a molecule, e.g., a contiguous stretch of nucleotides in an RNA.
  • the definition of “segment,” unless otherwise specifically defined in a particular context, is not limited to a specific number of total base pairs, and may include regions of RNA molecules that are of any total length and may or may not include regions with complementarity to other molecules.
  • guide RNA can be a single-molecule guide RNA or a two-molecule guide RNA.
  • guide RNA or “gRNA” is inclusive, referring both to two-molecule guide RNAs and to single-molecule guide RNAs (i.e., sgRNAs).
  • the variant Cas9 site-directed polypeptide comprises: i) an RNA-binding portion that interacts with the guide RNA; and an activity portion that exhibits reduced endodeoxyribonuclease activity.
  • the guide RNA and the variant Cas9 polypeptide form a complex in the host cell; the complex selectively modulates transcription of a target DNA in the host cell.
  • a transcription modulation method of the present disclosure provides for selective modulation (e.g., reduction or increase) of a target nucleic acid in a host cell.
  • selective modulation e.g., reduction or increase
  • “selective” reduction of transcription of a target nucleic acid reduces transcription of the target nucleic acid by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or greater than 90%, compared to the level of transcription of the target nucleic acid in the absence of a guide RNA/variant Cas9 polypeptide complex.
  • Selective reduction of transcription of a target nucleic acid reduces transcription of the target nucleic acid, but does not substantially reduce transcription of a non-target nucleic acid, e.g., transcription of a non-target nucleic acid is reduced, if at all, by less than 10% compared to the level of transcription of the non-target nucleic acid in the absence of the guide RNA/variant Cas9 polypeptide complex.
  • “Selective” increased transcription of a target DNA can increase transcription of the target DNA by at least about 1.1 fold (e.g., at least about 1.2 fold, at least about 1.3 fold, at least about 1.4 fold, at least about 1.5 fold, at least about 1.6 fold, at least about 1.7 fold, at least about 1.8 fold, at least about 1.9 fold, at least about 2 fold, at least about 2.5 fold, at least about 3 fold, at least about 3.5 fold, at least about 4 fold, at least about 4.5 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 12 fold, at least about 15 fold, or at least about 20-fold) compared to the level of transcription of the target DNA in the absence of a guide RNA/variant Cas9 polypeptide complex.
  • Selective increase of transcription of a target DNA increases transcription of the target DNA, but does not substantially increase transcription of a non-target DNA, e.g., transcription of a non-target DNA is increased, if at all, by less than about 5-fold (e.g., less than about 4-fold, less than about 3-fold, less than about 2-fold, less than about 1.8-fold, less than about 1.6-fold, less than about 1.4-fold, less than about 1.2-fold, or less than about 1.1-fold) compared to the level of transcription of the non-targeted DNA in the absence of the guide RNA/variant Cas9 polypeptide complex.
  • less than about 5-fold e.g., less than about 4-fold, less than about 3-fold, less than about 2-fold, less than about 1.8-fold, less than about 1.6-fold, less than about 1.4-fold, less than about 1.2-fold, or less than about 1.1-fold
  • increased transcription can be achieved by fusing dCas9 to a heterologous sequence.
  • Suitable fusion partners include, but are not limited to, a polypeptide that provides an activity that indirectly increases transcription by acting directly on the target DNA or on a polypeptide (e.g., a histone or other DNA-binding protein) associated with the target DNA.
  • Suitable fusion partners include, but are not limited to, a polypeptide that provides for methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, or demyristoylation activity.
  • Additional suitable fusion partners include, but are not limited to, a polypeptide that directly provides for increased transcription of the target nucleic acid (e.g., a transcription activator or a fragment thereof, a protein or fragment thereof that recruits a transcription activator, a small molecule/drug-responsive transcription regulator, etc.).
  • a polypeptide that directly provides for increased transcription of the target nucleic acid e.g., a transcription activator or a fragment thereof, a protein or fragment thereof that recruits a transcription activator, a small molecule/drug-responsive transcription regulator, etc.
  • a non-limiting example of a method using a dCas9 fusion protein to increase transcription in a prokaryote includes a modification of the bacterial one-hybrid (B1H) or two-hybrid (B2H) system.
  • B1H bacterial one-hybrid
  • B2H two-hybrid
  • AD bacterial transcription activation domain
  • a dCas9 can be fused to a heterologous sequence comprising an AD.
  • the AD e.g., RNAPa
  • the BD is not directly fused to the AD; instead, their interaction is mediated by a protein-protein interaction (e.g., GAL11P-GAL4 interaction).
  • dCas9 can be fused to a first protein sequence that provides for protein-protein interaction (e.g., the yeast GAL11P and/or GAL4 protein) and RNAa can be fused to a second protein sequence that completes the protein-protein interaction (e.g., GAL4 if GAL11P is fused to dCas9, GAL11P if GAL4 is fused to dCas9, etc.).
  • the binding affinity between GAL11P and GAL4 increases the efficiency of binding and transcription firing rate.
  • a non-limiting example of a method using a dCas9 fusion protein to increase transcription in a eukaryotes includes fusion of dCas9 to an activation domain (AD) (e.g., GAL4, herpesvirus activation protein VP16 or VP64, human nuclear factor NF- ⁇ B p65 subunit, etc.).
  • AD activation domain
  • expression of the dCas9 fusion protein can be controlled by an inducible promoter (e.g., Tet-ON, Tet-OFF, etc.).
  • the guide RNA can be design to target known transcription response elements (e.g., promoters, enhancers, etc.), known upstream activating sequences (UAS), sequences of unknown or known function that are suspected of being able to control expression of the target DNA, etc.
  • known transcription response elements e.g., promoters, enhancers, etc.
  • UAS upstream activating sequences
  • Non-limiting examples of fusion partners to accomplish increased or decreased transcription include, but are not limited to, transcription activator and transcription repressor domains (e.g., the Kriippel associated box (KRAB or SKD); the Mad mSIN3 interaction domain (SID); the ERF repressor domain (ERD), etc).
  • transcription activator and transcription repressor domains e.g., the Kriippel associated box (KRAB or SKD); the Mad mSIN3 interaction domain (SID); the ERF repressor domain (ERD), etc.
  • the dCas9 fusion protein is targeted by the guide RNA to a specific location (i.e., sequence) in the target DNA and exerts locus-specific regulation such as blocking RNA polymerase binding to a promoter (which selectively inhibits transcription activator function), and/or modifying the local chromatin status (e.g., when a fusion sequence is used that modifies the target DNA or modifies a polypeptide associated with the target DNA).
  • the changes are transient (e.g., transcription repression or activation).
  • the changes are inheritable (e.g., when epigenetic modifications are made to the target DNA or to proteins associated with the target DNA, e.g., nucleosomal histones).
  • the heterologous sequence can be fused to the C-terminus of the dCas9 polypeptide. In some embodiments, the heterologous sequence can be fused to the N-terminus of the dCas9 polypeptide. In some embodiments, the heterologous sequence can be fused to an internal portion (i.e., a portion other than the N- or C-terminus) of the dCas9 polypeptide.
  • the biological effects of a method using a dCas9 fusion protein can be detected by any convenient method (e.g., gene expression assays; chromatin-based assays, e.g., Chromatin immunoPrecipitation (ChiP), Chromatin in vivo Assay (CiA), etc.; and the like).
  • any convenient method e.g., gene expression assays; chromatin-based assays, e.g., Chromatin immunoPrecipitation (ChiP), Chromatin in vivo Assay (CiA), etc.; and the like).
  • a method involves use of two or more different guide RNAs.
  • two different guide RNAs can be used in a single host cell, where the two different guide RNAs target two different target sequences in the same target nucleic acid.
  • a transcriptional modulation method can further comprise introducing into the host cell a second guide RNA, or a nucleic acid comprising a nucleotide sequence encoding the second guide RNA, where the second guide RNA comprises: i) a first segment comprising a nucleotide sequence that is complementary to a second target sequence in the target DNA; ii) a second segment that interacts with the site-directed polypeptide; and iii) a transcriptional terminator.
  • use of two different guide RNAs targeting two different targeting sequences in the same target nucleic acid provides for increased modulation (e.g., reduction or increase) in transcription of the target nucleic acid.
  • a transcriptional modulation method can further comprise introducing into the host cell a second guide RNA, or a nucleic acid comprising a nucleotide sequence encoding the second guide RNA, where the second guide RNA comprises: i) a first segment comprising a nucleotide sequence that is complementary to a target sequence in at least a second target DNA; ii) a second segment that interacts with the site-directed polypeptide; and iii) a transcriptional terminator.
  • a nucleic acid comprises a modification or sequence that provides for an additional desirable feature (e.g., modified or regulated stability; subcellular targeting; tracking, e.g., a fluorescent label; a binding site for a protein or protein complex; etc.).
  • an additional desirable feature e.g., modified or regulated stability; subcellular targeting; tracking, e.g., a fluorescent label; a binding site for a protein or protein complex; etc.
  • Non-limiting examples include: a 5′ cap (e.g., a 7-methylguanylate cap (m 7 G)); a 3′ polyadenylated tail (i.e., a 3′ poly(A) tail); a riboswitch sequence or an aptamer sequence (e.g., to allow for regulated stability and/or regulated accessibility by proteins and/or protein complexes); a terminator sequence; a sequence that forms a dsRNA duplex (i.e., a hairpin)); a modification or sequence that targets the RNA to a subcellular location (e.g., nucleus, mitochondria, chloroplasts, and the like); a modification or sequence that provides for tracking (e.g., direct conjugation to a fluorescent molecule, conjugation to a moiety that facilitates fluorescent detection, a sequence that allows for fluorescent detection, etc.); a modification or sequence that provides a binding site for proteins (e.g., proteins that act on DNA, including transcriptional activators, transcriptional
  • the DNA-targeting segment (or “DNA-targeting sequence”) of a guide RNA comprises a nucleotide sequence that is complementary to a specific sequence within a target DNA (the complementary strand of the target DNA).
  • the DNA-targeting segment of a guide RNA interacts with a target DNA in a sequence-specific manner via hybridization (i.e., base pairing).
  • the nucleotide sequence of the DNA-targeting segment may vary and determines the location within the target DNA that the guide RNA and the target DNA will interact.
  • the DNA-targeting segment of a guide RNA can be modified (e.g., by genetic engineering) to hybridize to any desired sequence within a target DNA.
  • Stability Control Sequence e.g., Transcriptional Terminator Segment
  • a stability control sequence influences the stability of an RNA (e.g., a guide RNA, a targeter-RNA, an activator-RNA, etc.).
  • RNA e.g., a guide RNA, a targeter-RNA, an activator-RNA, etc.
  • a suitable stability control sequence is a transcriptional terminator segment (i.e., a transcription termination sequence).
  • a transcriptional terminator segment of a guide RNA can have a total length of from about 10 nucleotides to about 100 nucleotides, e.g., from about 10 nucleotides (nt) to about 20 nt, from about 20 nt to about 30 nt, from about 30 nt to about 40 nt, from about 40 nt to about 50 nt, from about 50 nt to about 60 nt, from about 60 nt to about 70 nt, from about 70 nt to about 80 nt, from about 80 nt to about 90 nt, or from about 90 nt to about 100 nt.
  • the transcriptional terminator segment can have a length of from about 15 nucleotides (nt) to about 80 nt, from about 15 nt to about 50 nt, from about 15 nt to about 40 nt, from about 15 nt to about 30 nt or from about 15 nt to about 25 nt.
  • the transcription termination sequence is one that is functional in a eukaryotic cell. In some cases, the transcription termination sequence is one that is functional in a prokaryotic cell.
  • Nucleotide sequences that can be included in a stability control sequence include, for example, 5′-UAAUCCCACAGCCGCCAGUUCCGCUGGCGGCAUUUU-5′ (a Rho-independent trp termination site).
  • a guide RNA comprises at least one additional segment at either the 5′ or 3′ end.
  • a suitable additional segment can comprise a 5′ cap (e.g., a 7-methylguanylate cap (m 7 G)); a 3′ polyadenylated tail (i.e., a 3′ poly(A) tail); a riboswitch sequence (e.g., to allow for regulated stability and/or regulated accessibility by proteins and protein complexes); a sequence that forms a dsRNA duplex (i.e., a hairpin)); a sequence that targets the RNA to a subcellular location (e.g., nucleus, mitochondria, chloroplasts, and the like); a modification or sequence that provides for tracking (e.g., direct conjugation to a fluorescent molecule, conjugation to a moiety that facilitates fluorescent detection, a sequence that allows for fluorescent detection, etc.); a modification or sequence that provides a binding site for proteins (e.g., proteins that
  • multiple guide RNAs are used simultaneously in the same cell to simultaneously modulate transcription at different locations on the same target DNA or on different target DNAs.
  • two or more guide RNAs target the same gene or transcript or locus.
  • two or more guide RNAs target different unrelated loci.
  • two or more guide RNAs target different, but related loci.
  • the guide RNAs are small and robust they can be simultaneously present on the same expression vector and can even be under the same transcriptional control if so desired.
  • two or more (e.g., 3 or more, 4 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, or 50 or more) guide RNAs are simultaneously expressed in a target cell (from the same or different vectors).
  • the expressed guide RNAs can be differently recognized by Cas9 proteins from different bacteria, such as S. pyogenes, S. thermophilus, L. innocua , and N. meningitidis.
  • multiple guide RNAs can be encoded in an array mimicking naturally occurring CRISPR arrays of targeter RNAs and corresponding tracrRNAs (activator RNAs).
  • the targeting segments are encoded as approximately 30 nucleotide long sequences (can be about 16 to about 100 nt) and are separated by CRISPR repeat sequences.
  • the array and tracrRNAs are introduced to a cell by DNAs encoding the RNAs. In some cases, they are introduced to the cell as RNAs.
  • an artificial RNA processing system mediated by the Csy4 endoribonuclease can be used.
  • Multiple guide RNAs can be concatenated into a tandem array on a precursor transcript (e.g., expressed from a U6 promoter), and separated by Csy4-specific RNA sequence.
  • Co-expressed Csy4 protein cleaves the precursor transcript into multiple guide RNAs.
  • Advantages for using an RNA processing system include: first, there is no need to use multiple promoters; second, since all guide RNAs are processed from a precursor transcript, their concentrations are normalized for similar dCas9-binding.
  • Csy4 is a small endoribonuclease (RNase) protein derived from bacteria Pseudomonas aeruginosa .
  • RNase Ribonuclease
  • Csy4 specifically recognizes a minimal 17-bp RNA hairpin, and exhibits rapid ( ⁇ 1 min) and highly efficient (>99.9%) RNA cleavage. Unlike most RNases, the cleaved RNA fragment remains stable and functionally active.
  • the Csy4-based RNA cleavage can be repurposed into an artificial RNA processing system. In this system, the 17-bp RNA hairpins are inserted between multiple RNA fragments that are transcribed as a precursor transcript from a single promoter. Co-expression of Csy4 is effective in generating individual RNA fragments.
  • a guide RNA and a variant Cas9 site-directed polypeptide form a complex.
  • the guide RNA provides target specificity to the complex by comprising a nucleotide sequence that is complementary to a sequence of a target DNA.
  • the variant Cas9 site-directed polypeptide has reduced endodeoxyribonuclease activity.
  • a variant Cas9 site-directed polypeptide suitable for use in a transcription modulation method of the present disclosure exhibits less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1%, or less than about 0.1%, of the endodeoxyribonuclease activity of a wild-type Cas9 polypeptide, e.g., a wild-type Cas9 polypeptide comprising an amino acid sequence set out in SEQ ID NO:8.
  • the variant Cas9 site-directed polypeptide has substantially no detectable endodeoxyribonuclease activity.
  • a site-directed polypeptide has reduced catalytic activity (e.g., when a SEQ ID NO: 8 S. pyogenes Cas9 protein has a D10, G12, G17, E762, H840, N863, H982, H983, A984, D986, and/or a A987 mutation, e.g., D10A, G12A, G17A, E762A, H840A, N863A, H982A, H983A, A984A, and/or D986A)
  • the polypeptide can still bind to target DNA in a site-specific manner (because it is still guided to a target DNA sequence by a guide RNA) as long as it retains the ability to interact with the guide RNA.
  • a suitable variant Cas9 site-directed polypeptide comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100% amino acid sequence identity to amino acids 7-166 and/or 731-1003 of SEQ ID NO: 8, or to the corresponding portions in any one of the amino acid sequences SEQ ID NOs: 1-800.
  • the variant Cas9 site-directed polypeptide is a nickase that can cleave the complementary strand of the target DNA but has reduced ability to cleave the non-complementary strand of the target DNA.
  • the variant Cas9 site-directed polypeptide can have a mutation (amino acid substitution) that reduces the function of the RuvC domain.
  • the variant Cas9 site-directed polypeptide is a D10A (aspartate to alanine) mutation of SEQ ID NO: 8 (or the corresponding mutation of any of the amino acid sequences set forth in SEQ ID NOs: 1-800).
  • the variant Cas9 site-directed polypeptide in a nickase that can cleave the non-complementary strand of the target DNA but has reduced ability to cleave the complementary strand of the target DNA.
  • the variant Cas9 site-directed polypeptide can have a mutation (amino acid substitution) that reduces the function of the HNH domain (RuvC/HNH/RuvC domain motifs, “domain 2”).
  • the variant Cas9 site-directed polypeptide is a H840A (histidine to alanine at amino acid position 840 of SEQ ID NO:8) or the corresponding mutation of any of the amino acid sequences set forth in SEQ ID NOs: 1-800).
  • the variant Cas9 site-directed polypeptide has a reduced ability to cleave both the complementary and the non-complementary strands of the target DNA.
  • the variant Cas9 site-directed polypeptide harbors both D10A and H840A mutations of SEQ ID NO: 8 (or the corresponding mutations of any of the amino acid sequences set forth in SEQ ID NOs: 1-800). Other residues can be mutated to achieve the same effect (i.e. inactivate one or the other nuclease portions).
  • Cas9 residues D10, G12, G17, E762, H840, N863, H982, H983, A984, D986, and/or A987 of SEQ ID NO: 8 can be altered (i.e., substituted) (see Table 1 for examples of the conservation of Cas9 amino acid residues). Also, mutations other than alanine substitutions are contemplated.
  • a variant Cas9 endonuclease comprises one or more mutations corresponding to a S. pyogenes Cas9 mutation E762A, HH983AA or D986A in SEQ ID NO: 8.
  • the modified Cas 9 endonuclease further comprises one or more mutations corresponding to a S. pyogenes Cas9 mutation D10A, H840A, G12A, G17A, N854A, N863A, N982A or A984A in SEQ ID NO: 8.
  • the variant Cas9 site-directed polypeptide is a fusion polypeptide (a “variant Cas9 fusion polypeptide”), i.e., a fusion polypeptide comprising: i) a variant Cas9 site-directed polypeptide; and ii) a covalently linked heterologous polypeptide (also referred to as a “fusion partner”).
  • variant Cas9 fusion polypeptide a fusion polypeptide
  • a fusion polypeptide comprising: i) a variant Cas9 site-directed polypeptide; and ii) a covalently linked heterologous polypeptide (also referred to as a “fusion partner”).
  • the heterologous polypeptide may exhibit an activity (e.g., enzymatic activity) that will also be exhibited by the variant Cas9 fusion polypeptide (e.g., methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitinating activity, etc.).
  • a heterologous nucleic acid sequence may be linked to another nucleic acid sequence (e.g., by genetic engineering) to generate a chimeric nucleotide sequence encoding a chimeric polypeptide.
  • a variant Cas9 fusion polypeptide is generated by fusing a variant Cas9 polypeptide with a heterologous sequence that provides for subcellular localization (i.e., the heterologous sequence is a subcellular localization sequence, e.g., a nuclear localization signal (NLS) for targeting to the nucleus; a mitochondrial localization signal for targeting to the mitochondria; a chloroplast localization signal for targeting to a chloroplast; an ER retention signal; and the like).
  • a subcellular localization sequence e.g., a nuclear localization signal (NLS) for targeting to the nucleus; a mitochondrial localization signal for targeting to the mitochondria; a chloroplast localization signal for targeting to a chloroplast; an ER retention signal; and the like.
  • the heterologous sequence can provide a tag (i.e., the heterologous sequence is a detectable label) for ease of tracking and/or purification (e.g., a fluorescent protein, e.g., green fluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato, and the like; a histidine tag, e.g., a 6 ⁇ His tag; a hemagglutinin (HA) tag; a FLAG tag; a Myc tag; and the like).
  • a fluorescent protein e.g., green fluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato, and the like
  • GFP green fluorescent protein
  • YFP green fluorescent protein
  • RFP red fluorescent protein
  • CFP CFP
  • mCherry mCherry
  • tdTomato e.g., a histidine tag
  • HA hemagglutinin
  • the heterologous sequence can provide for increased or decreased stability (i.e., the heterologous sequence is a stability control peptide, e.g., a degron, which in some cases is controllable (e.g., a temperature sensitive or drug controllable degron sequence, see below).
  • a stability control peptide e.g., a degron
  • controllable e.g., a temperature sensitive or drug controllable degron sequence, see below.
  • the heterologous sequence can provide for increased or decreased transcription from the target DNA (i.e., the heterologous sequence is a transcription modulation sequence, e.g., a transcription factor/activator or a fragment thereof, a protein or fragment thereof that recruits a transcription factor/activator, a transcription repressor or a fragment thereof, a protein or fragment thereof that recruits a transcription repressor, a small molecule/drug-responsive transcription regulator, etc.).
  • a transcription modulation sequence e.g., a transcription factor/activator or a fragment thereof, a protein or fragment thereof that recruits a transcription factor/activator, a transcription repressor or a fragment thereof, a protein or fragment thereof that recruits a transcription repressor, a small molecule/drug-responsive transcription regulator, etc.
  • the heterologous sequence can provide a binding domain (i.e., the heterologous sequence is a protein binding sequence, e.g., to provide the ability of a chimeric dCas9 polypeptide to bind to another protein of interest, e.g., a DNA or histone modifying protein, a transcription factor or transcription repressor, a recruiting protein, etc.).
  • a protein binding sequence e.g., to provide the ability of a chimeric dCas9 polypeptide to bind to another protein of interest, e.g., a DNA or histone modifying protein, a transcription factor or transcription repressor, a recruiting protein, etc.
  • Suitable fusion partners that provide for increased or decreased stability include, but are not limited to degron sequences.
  • Degrons are readily understood by one of ordinary skill in the art to be amino acid sequences that control the stability of the protein of which they are part. For example, the stability of a protein comprising a degron sequence is controlled at least in part by the degron sequence.
  • a suitable degron is constitutive such that the degron exerts its influence on protein stability independent of experimental control (i.e., the degron is not drug inducible, temperature inducible, etc.)
  • the degron provides the variant Cas9 polypeptide with controllable stability such that the variant Cas9 polypeptide can be turned “on” (i.e., stable) or “off” (i.e., unstable, degraded) depending on the desired conditions.
  • the variant Cas9 polypeptide may be functional (i.e., “on”, stable) below a threshold temperature (e.g., 42° C., 41° C., 40° C., 39° C., 38° C., 37° C., 36° C., 35° C., 34° C., 33° C., 32° C., 31° C., 30° C., etc.) but non-functional (i.e., “off”, degraded) above the threshold temperature.
  • a threshold temperature e.g., 42° C., 41° C., 40° C., 39° C., 38° C., 37° C., 36° C., 35° C., 34° C., 33° C., 32° C., 31° C., 30° C., etc.
  • non-functional i.e., “off”, degraded
  • the degron is a drug inducible degron
  • the presence or absence of drug can switch the protein from an “off” (i.e., unstable) state to an “on” (i.e., stable) state or vice versa.
  • An exemplary drug inducible degron is derived from the FKBP12 protein. The stability of the degron is controlled by the presence or absence of a small molecule that binds to the degron.
  • suitable degrons include, but are not limited to those degrons controlled by Shield-1, DHFR, auxins, and/or temperature.
  • suitable degrons are known in the art (e.g., Dohmen et al., Science, 1994. 263(5151): p. 1273-1276: Heat-inducible degron: a method for constructing temperature-sensitive mutants; Schoeber et al., Am J Physiol Renal Physiol. 2009 January; 296(1):F204-11: Conditional fast expression and function of multimeric TRPV5 channels using Shield-1; Chu et al., Bioorg Med Chem Lett.
  • Exemplary degron sequences have been well-characterized and tested in both cells and animals. Thus, fusing Cas9 to a degron sequence produces a “tunable” and “inducible” Cas9 polypeptide.
  • Any of the fusion partners described herein can be used in any desirable combination.
  • a Cas9 fusion protein can comprise a YFP sequence for detection, a degron sequence for stability, and transcription activator sequence to increase transcription of the target DNA.
  • the number of fusion partners that can be used in a Cas9 fusion protein is unlimited.
  • a Cas9 fusion protein comprises one or more (e.g. two or more, three or more, four or more, or five or more) heterologous sequences.
  • Suitable fusion partners include, but are not limited to, a polypeptide that provides for methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, or demyristoylation activity, any of which can be directed at modifying the DNA directly (e.g., methylation of DNA) or at modifying a DNA-associated polypeptide (e.g., a histone or DNA binding protein).
  • a polypeptide that provides for methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase
  • fusion partners include, but are not limited to boundary elements (e.g., CTCF), proteins and fragments thereof that provide periphery recruitment (e.g., Lamin A, Lamin B, etc.), and protein docking elements (e.g., FKBP/FRB, Pil 1/Aby 1, etc.).
  • boundary elements e.g., CTCF
  • proteins and fragments thereof that provide periphery recruitment e.g., Lamin A, Lamin B, etc.
  • protein docking elements e.g., FKBP/FRB, Pil 1/Aby 1, etc.
  • a site-directed modifying polypeptide can be codon-optimized. This type of optimization is known in the art and entails the mutation of foreign-derived DNA to mimic the codon preferences of the intended host organism or cell while encoding the same protein. Thus, the codons are changed, but the encoded protein remains unchanged. For example, if the intended target cell was a human cell, a human codon-optimized dCas9 (or dCas9 variant) would be a suitable site-directed modifying polypeptide.
  • a mouse codon-optimized Cas9 or variant, e.g., enzymatically inactive variant
  • a suitable Cas9 site-directed polypeptide While codon optimization is not required, it is acceptable and may be preferable in certain cases.
  • Polyadenylation signals can also be chosen to optimize expression in the intended host.
  • a method of the present disclosure to modulate transcription may be employed to induce transcriptional modulation in mitotic or post-mitotic cells in vivo and/or ex vivo and/or in vitro.
  • a mitotic and/or post-mitotic cell can be any of a variety of host cell, where suitable host cells include, but are not limited to, a bacterial cell; an archaeal cell; a single-celled eukaryotic organism; a plant cell; an algal cell, e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C.
  • a fungal cell e.g., an insect, a cnidarian, an echinoderm, a nematode, etc.
  • a eukaryotic parasite e.g., a malarial parasite, e.g., Plasmodium fakiparum ; a helminth; etc.
  • a cell from a vertebrate animal e.g., fish, amphibian, reptile, bird, mammal
  • a mammalian cell e.g., a rodent cell, a human cell, a non-human primate cell, etc.
  • Suitable host cells include naturally-occurring cells; genetically modified cells (e.g., cells genetically modified in a laboratory, e.g., by the “hand of man”); and cells manipulated in vitro in any way. In some cases, a host cell is isolated.
  • a stem cell e.g. an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell, a germ cell; a somatic cell, e.g. a fibroblast, a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell; an in vitro or in vivo embryonic cell of an embryo at any stage, e.g., a 1-cell, 2-cell, 4-cell, 8-cell, etc. stage zebrafish embryo; etc.).
  • ES embryonic stem
  • iPS induced pluripotent stem
  • a germ cell e.g. a somatic cell, e.g. a fibroblast, a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell
  • an in vitro or in vivo embryonic cell of an embryo at any stage e
  • Cells may be from established cell lines or they may be primary cells, where “primary cells”, “primary cell lines”, and “primary cultures” are used interchangeably herein to refer to cells and cells cultures that have been derived from a subject and allowed to grow in vitro for a limited number of passages, i.e. splittings, of the culture.
  • primary cultures include cultures that may have been passaged 0 times, 1 time, 2 times, 4 times, 5 times, 10 times, or 15 times, but not enough times go through the crisis stage.
  • Primary cell lines can be are maintained for fewer than 10 passages in vitro.
  • Target cells are in many embodiments unicellular organisms, or are grown in culture.
  • the cells may be harvest from an individual by any convenient method.
  • leukocytes may be conveniently harvested by apheresis, leukocytapheresis, density gradient separation, etc., while cells from tissues such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach, etc. are most conveniently harvested by biopsy.
  • An appropriate solution may be used for dispersion or suspension of the harvested cells.
  • Such solution will generally be a balanced salt solution, e.g.
  • fetal calf serum or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration, e.g., from 5-25 mM.
  • Convenient buffers include HEPES, phosphate buffers, lactate buffers, etc.
  • the cells may be used immediately, or they may be stored, frozen, for long periods of time, being thawed and capable of being reused.
  • the cells will usually be frozen in 10% dimethyl sulfoxide (DMSO), 50% serum, 40% buffered medium, or some other such solution as is commonly used in the art to preserve cells at such freezing temperatures, and thawed in a manner as commonly known in the art for thawing frozen cultured cells.
  • DMSO dimethyl sulfoxide
  • a guide RNA, or a nucleic acid comprising a nucleotide sequence encoding same can be introduced into a host cell by any of a variety of well-known methods.
  • a method involves introducing into a host cell a nucleic acid comprising a nucleotide sequence encoding a variant Cas9 site-directed polypeptide, such a nucleic acid can be introduced into a host cell by any of a variety of well-known methods.
  • nucleic acid e.g., an expression construct
  • Suitable methods include, include e.g., viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, nanoparticle-mediated nucleic acid delivery (see, e.g., Panyam et., al Adv Drug Deliv Rev. 2012 Sep. 13. pii: 50169-409X(12)00283-9. doi: 10.1016/j.addr.2012.09.023), and the like.
  • PKI polyethyleneimine
  • the present disclosure provides an isolated nucleic acid comprising a nucleotide sequence encoding a guide RNA.
  • a nucleic acid also comprises a nucleotide sequence encoding a variant Cas9 site-directed polypeptide.
  • a method involves introducing into a host cell (or a population of host cells) one or more nucleic acids comprising nucleotide sequences encoding a guide RNA and/or a variant Cas9 site-directed polypeptide.
  • a cell comprising a target DNA is in vitro.
  • a cell comprising a target DNA is in vivo.
  • Suitable nucleic acids comprising nucleotide sequences encoding a guide RNA and/or a site-directed polypeptide include expression vectors, where an expression vector comprising a nucleotide sequence encoding a guide RNA and/or a site-directed polypeptide is a “recombinant expression vector.”
  • the recombinant expression vector is a viral construct, e.g., a recombinant adeno-associated virus construct (see, e.g., U.S. Pat. No. 7,078,387), a recombinant adenoviral construct, a recombinant lentiviral construct, a recombinant retroviral construct, etc.
  • Suitable expression vectors include, but are not limited to, viral vectors (e.g.
  • viral vectors based on vaccinia virus; poliovirus; adenovirus see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther
  • SV40 herpes simplex virus
  • human immunodeficiency virus see, e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816, 1999
  • a retroviral vector e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus
  • retroviral vector e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myelop
  • Suitable expression vectors are known to those of skill in the art, and many are commercially available.
  • the following vectors are provided by way of example; for eukaryotic host cells: pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia).
  • any other vector may be used so long as it is compatible with the host cell.
  • any of a number of suitable transcription and translation control elements including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector (see e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544).
  • a nucleotide sequence encoding a guide RNA and/or a variant Cas9 site-directed polypeptide is operably linked to a control element, e.g., a transcriptional control element, such as a promoter.
  • a control element e.g., a transcriptional control element, such as a promoter.
  • the transcriptional control element may be functional in either a eukaryotic cell, e.g., a mammalian cell; or a prokaryotic cell (e.g., bacterial or archaeal cell).
  • a nucleotide sequence encoding a guide RNA and/or a variant Cas9 site-directed polypeptide is operably linked to multiple control elements that allow expression of the nucleotide sequence encoding a guide RNA and/or a variant Cas9 site-directed polypeptide in both prokaryotic and eukaryotic cells.
  • a promoter can be a constitutively active promoter (i.e., a promoter that is constitutively in an active/“ON” state), it may be an inducible promoter (i.e., a promoter whose state, active/“ON” or inactive/“OFF”, is controlled by an external stimulus, e.g., the presence of a particular temperature, compound, or protein.), it may be a spatially restricted promoter (i.e., transcriptional control element, enhancer, etc.)(e.g., tissue specific promoter, cell type specific promoter, etc.), and it may be a temporally restricted promoter (i.e., the promoter is in the “ON” state or “OFF” state during specific stages of embryonic development or during specific stages of a biological process, e.g., hair follicle cycle in mice).
  • a constitutively active promoter i.e., a promoter that is constitutively in an active/“ON” state
  • it may be an inducible promote
  • Suitable promoters can be derived from viruses and can therefore be referred to as viral promoters, or they can be derived from any organism, including prokaryotic or eukaryotic organisms. Suitable promoters can be used to drive expression by any RNA polymerase (e.g., pol I, pol II, pol III).
  • RNA polymerase e.g., pol I, pol II, pol III
  • Exemplary promoters include, but are not limited to the SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, a human U6 small nuclear promoter (U6) (Miyagishi et al., Nature Biotechnology 20, 497-500 (2002)), an enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep. 1; 31(17)), a human H1 promoter (H1), and the like.
  • LTR mouse mammary tumor virus long terminal repeat
  • Ad MLP adenovirus major late promoter
  • HSV herpes simplex virus
  • CMV cytomegalovirus
  • CMVIE C
  • inducible promoters include, but are not limited to T7 RNA polymerase promoter, T3 RNA polymerase promoter, Isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated promoter, lactose induced promoter, heat shock promoter, Tetracycline-regulated promoter (e.g., Tet-ON, Tet-OFF, etc.), Steroid-regulated promoter, Metal-regulated promoter, estrogen receptor-regulated promoter, etc.
  • Inducible promoters can therefore be regulated by molecules including, but not limited to, doxycycline; RNA polymerase, e.g., T7 RNA polymerase; an estrogen receptor; an estrogen receptor fusion; etc.
  • the promoter is a spatially restricted promoter (i.e., cell type specific promoter, tissue specific promoter, etc.) such that in a multi-cellular organism, the promoter is active (i.e., “ON”) in a subset of specific cells.
  • spatially restricted promoters may also be referred to as enhancers, transcriptional control elements, control sequences, etc.
  • any convenient spatially restricted promoter may be used and the choice of suitable promoter (e.g., a brain specific promoter, a promoter that drives expression in a subset of neurons, a promoter that drives expression in the germline, a promoter that drives expression in the lungs, a promoter that drives expression in muscles, a promoter that drives expression in islet cells of the pancreas, etc.) will depend on the organism.
  • various spatially restricted promoters are known for plants, flies, worms, mammals, mice, etc.
  • a spatially restricted promoter can be used to regulate the expression of a nucleic acid encoding a site-directed polypeptide in a wide variety of different tissues and cell types, depending on the organism.
  • Some spatially restricted promoters are also temporally restricted such that the promoter is in the “ON” state or “OFF” state during specific stages of embryonic development or during specific stages of a biological process (e.g., hair follicle cycle in mice).
  • spatially restricted promoters include, but are not limited to, neuron-specific promoters, adipocyte-specific promoters, cardiomyocyte-specific promoters, smooth muscle-specific promoters, photoreceptor-specific promoters, etc.
  • Neuron-specific spatially restricted promoters include, but are not limited to, a neuron-specific enolase (NSE) promoter (see, e.g., EMBL HSENO2, X51956); an aromatic amino acid decarboxylase (AADC) promoter; a neurofilament promoter (see, e.g., GenBank HUMNFL, L04147); a synapsin promoter (see, e.g., GenBank HUMSYNIB, M55301); a thy-1 promoter (see, e.g., Chen et al. (1987) Cell 51:7-19; and Llewellyn, et al. (2010) Nat. Med.
  • NSE neuron-specific enolase
  • AADC aromatic amino acid decarboxylase
  • Adipocyte-specific spatially restricted promoters include, but are not limited to aP2 gene promoter/enhancer, e.g., a region from ⁇ 5.4 kb to +21 bp of a human aP2 gene (see, e.g., Tozzo et al. (1997) Endocrinol. 138:1604; Ross et al. (1990) Proc. Natl. Acad. Sci. USA 87:9590; and Pavjani et al. (2005) Nat. Med. 11:797); a glucose transporter-4 (GLUT4) promoter (see, e.g., Knight et al. (2003) Proc. Natl. Acad. Sci.
  • aP2 gene promoter/enhancer e.g., a region from ⁇ 5.4 kb to +21 bp of a human aP2 gene (see, e.g., Tozzo et al. (1997) Endocrinol. 138:160
  • fatty acid translocase (FAT/CD36) promoter see, e.g., Kuriki et al. (2002) Biol. Pharm. Bull. 25:1476; and Sato et al. (2002) J. Biol. Chem. 277:15703
  • SCD1 stearoyl-CoA desaturase-1
  • SCD1 stearoyl-CoA desaturase-1 promoter
  • leptin promoter see, e.g., Mason et al. (1998) Endocrinol. 139:1013; and Chen et al. (1999) Biochem. Biophys. Res. Comm.
  • adiponectin promoter see, e.g., Kita et al. (2005) Biochem. Biophys. Res. Comm. 331:484; and Chakrabarti (2010) Endocrinol. 151:2408
  • an adipsin promoter see, e.g., Platt et al. (1989) Proc. Natl. Acad. Sci. USA 86:7490
  • a resistin promoter see, e.g., Seo et al. (2003) Molec. Endocrinol. 17:1522); and the like.
  • Cardiomyocyte-specific spatially restricted promoters include, but are not limited to control sequences derived from the following genes: myosin light chain-2, a-myosin heavy chain, AE3, cardiac troponin C, cardiac actin, and the like.
  • Franz et al. (1997) Cardiovasc. Res. 35:560-566; Robbins et al. (1995) Ann. N.Y. Acad. Sci. 752:492-505; Linn et al. (1995) Circ. Res. 76:584591; Parmacek et al. (1994) Mol. Cell. Biol. 14:1870-1885; Hunter et al. (1993) Hypertension 22:608-617; and Sartorelli et al. (1992) Proc. Natl. Acad. Sci. USA 89:4047-4051.
  • Smooth muscle-specific spatially restricted promoters include, but are not limited to an SM22a promoter (see, e.g., Akyilrek et al. (2000) Mol. Med. 6:983; and U.S. Pat. No. 7,169,874); a smoothelin promoter (see, e.g., WO 2001/018048); an a-smooth muscle actin promoter; and the like.
  • a 0.4 kb region of the SM22a promoter, within which lie two CArG elements has been shown to mediate vascular smooth muscle cell-specific expression (see, e.g., Kim, et al. (1997) Mol. Cell. Biol. 17, 2266-2278; Li, et al., (1996) J. Cell Biol. 132, 849-859; and Moessler, et al. (1996) Development 122, 2415-2425).
  • Photoreceptor-specific spatially restricted promoters include, but are not limited to, a rhodopsin promoter; a rhodopsin kinase promoter (Young et al. (2003) Ophthalmol. Vis. Sci. 44:4076); a beta phosphodiesterase gene promoter (Nicoud et al. (2007) J. Gene Med. 9:1015); a retinitis pigmentosa gene promoter (Nicoud et al. (2007) supra); an interphotoreceptor retinoid-binding protein (IRBP) gene enhancer (Nicoud et al. (2007) supra); an IRBP gene promoter (Yokoyama et al. (1992) Exp Eye Res. 55:225); and the like.
  • a rhodopsin promoter a rhodopsin kinase promoter
  • a beta phosphodiesterase gene promoter Necoud et al. (2007) J. Gene
  • the present disclosure provides a library of guide RNAs.
  • the present disclosure provides a library of nucleic acids comprising nucleotides encoding guide RNAs.
  • a library of nucleic acids comprising nucleotides encoding guide RNAs can comprises a library of recombinant expression vectors comprising nucleotides encoding the guide RNAs.
  • a library can comprise from about 10 individual members to about 10 12 individual members; e.g., a library can comprise from about 10 individual members to about 10 2 individual members, from about 10 2 individual members to about 10 3 individual members, from about 10 3 individual members to about 10 5 individual members, from about 10 5 individual members to about 10 7 individual members, from about 10 7 individual members to about 10 9 individual members, or from about 10 9 individual members to about 10 12 individual members.
  • each individual member of a library differs from other members of the library in the nucleotide sequence of the DNA targeting segment of the guide RNA.
  • each individual member of a library can comprise the same or substantially the same nucleotide sequence of the protein-binding segment as all other members of the library; and can comprise the same or substantially the same nucleotide sequence of the transcriptional termination segment as all other members of the library; but differs from other members of the library in the nucleotide sequence of the DNA targeting segment of the guide RNA.
  • the library can comprise members that bind to different target nucleic acids.
  • a method for modulating transcription according to the present disclosure finds use in a variety of applications, which are also provided.
  • Applications include research applications; diagnostic applications; industrial applications; and treatment applications.
  • Research applications include, e.g., determining the effect of reducing or increasing transcription of a target nucleic acid on, e.g., development, metabolism, expression of a downstream gene, and the like.
  • High through-put genomic analysis can be carried out using a transcription modulation method, in which only the DNA-targeting segment of the guide RNA needs to be varied, while the protein-binding segment and the transcription termination segment can (in some cases) be held constant.
  • a library e.g., a library
  • a library comprising a plurality of nucleic acids used in the genomic analysis would include: a promoter operably linked to a guide RNA-encoding nucleotide sequence, where each nucleic acid would include a different DNA-targeting segment, a common protein-binding segment, and a common transcription termination segment.
  • a chip could contain over 5 ⁇ 10 4 unique guide RNAs. Applications would include large-scale phenotyping, gene-to-function mapping, and meta-genomic analysis.
  • the methods disclosed herein find use in the field of metabolic engineering. Because transcription levels can be efficiently and predictably controlled by designing an appropriate guide RNA, as disclosed herein, the activity of metabolic pathways (e.g., biosynthetic pathways) can be precisely controlled and tuned by controlling the level of specific enzymes (e.g., via increased or decreased transcription) within a metabolic pathway of interest. Metabolic pathways of interest include those used for chemical (fine chemicals, fuel, antibiotics, toxins, agonists, antagonists, etc.) and/or drug production.
  • Biosynthetic pathways of interest include but are not limited to (1) the mevalonate pathway (e.g., HMG-CoA reductase pathway) (converts acetyl-CoA to dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP), which are used for the biosynthesis of a wide variety of biomolecules including terpenoids/isoprenoids), (2) the non-mevalonate pathway (i.e., the “2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose 5-phosphate pathway” or “MEP/DOXP pathway” or “DXP pathway”)(also produces DMAPP and IPP, instead by converting pyruvate and glyceraldehyde 3-phosphate into DMAPP and IPP via an alternative pathway to the mevalonate pathway), (3) the polyketide synthesis pathway (produces a variety of polyketides via a variety of polyketide synth
  • Polyketides include naturally occurring small molecules used for chemotherapy (e. g., tetracyclin, and macrolides) and industrially important polyketides include rapamycin (immunosuppressant), erythromycin (antibiotic), lovastatin (anticholesterol drug), and epothilone B (anticancer drug)), (4) fatty acid synthesis pathways, (5) the DAHP (3-deoxy-D-arabino-heptulosonate 7-phosphate) synthesis pathway, (6) pathways that produce potential biofuels (such as short-chain alcohols and alkane, fatty acid methyl esters and fatty alcohols, isoprenoids, etc.), etc.
  • rapamycin immunosuppressant
  • erythromycin antibiotic
  • lovastatin anticholesterol drug
  • epothilone B anticancer drug
  • RNA/variant Cas9 site-directed polypeptide may be used to control (i.e., modulate, e.g., increase, decrease) the expression of another DNA-targeting RNA or another variant Cas9 site-directed polypeptide.
  • a first guide RNA may be designed to target the modulation of transcription of a second chimeric dCas9 polypeptide with a function that is different than the first variant Cas9 site-directed polypeptide (e.g., methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, etc.).
  • the second chimeric dCas9 polypeptide can be derived from a different species than the first dCas9 polypeptide above.
  • the second chimeric dCas9 polypeptide can be selected such that it may not interact with the first guide RNA. In other cases, the second chimeric dCas9 polypeptide can be selected such that it does interact with the first guide RNA. In some such cases, the activities of the two (or more) dCas9 proteins may compete (e.g., if the polypeptides have opposing activities) or may synergize (e.g., if the polypeptides have similar or synergistic activities).
  • any of the complexes in the network can be designed to control other guide RNAs or dCas9 polypeptides.
  • a guide RNA and variant Cas9 site-directed polypeptide can be targeted to any desired DNA sequence, the methods described herein can be used to control and regulate the expression of any desired target.
  • the integrated networks i.e., cascades of interactions
  • the level of expression of one component of the network may affect the level of expression (e.g., may increase or decrease the expression) of another component of the network.
  • the expression of one component may affect the expression of a different component in the same network, and the network may include a mix of components that increase the expression of other components, as well as components that decrease the expression of other components.
  • level of expression of one component may affect the level of expression of one or more different component(s) are for illustrative purposes, and are not limiting.
  • An additional layer of complexity may be optionally introduced into a network when one or more components are modified (as described above) to be manipulable (i.e., under experimental control, e.g., temperature control; drug control, i.e., drug inducible control; light control; etc.).
  • a first guide RNA can bind to the promoter of a second guide RNA, which controls the expression of a target therapeutic/metabolic gene.
  • conditional expression of the first guide RNA indirectly activates the therapeutic/metabolic gene.
  • RNA cascades of this type are useful, for example, for easily converting a repressor into an activator, and can be used to control the logics or dynamics of expression of a target gene.
  • a transcription modulation method can also be used for drug discovery and target validation.
  • Example 1 relates to Cas9 orthologs
  • Example 2 elates to exchangeability of bacterial RNase III enzymes
  • Example 3 relates to the Cas9 HNH and RuvC domains
  • Example 4 relates to exchangeability of Cas9 endonucleases in tracrRNA-directed pre-crRNA maturation by RNase III
  • Example 5 relates to PAMs of Cas9 orthologs
  • Example 6 relates to exchangeability of guide RNA and Cas9 endonucleases.
  • Supplementary Table S1 lists bacterial strains used in this study.
  • S. pyogenes, Streptococcus mutans, Campylobacter jejuni, N. meningitidis, Escherichia coli and Francisella novicida were grown as previously described (15,16).
  • BHI Brain Heart Infusion, Becton Dickinson
  • agar and BHI broth medium supplemented with 1% glucose and 1% lactose were used to culture S. thermophilus at 42° C. in a 5% CO 2 environment (16).
  • Pasteurella multocida and Staphylococcus aureus were grown at 37° C. on BHI agar plates and in BHI broth with shaking. Cell growth was monitored by measuring the optical density of cultures at 620 nm (OD 620 ) using a microplate reader (BioTek PowerWave).
  • E. coli was transformed with plasmid DNA according to standard protocols (35). Transformation of S. pyogenes was performed as previously described (36) with some modifications. S. pyogenes pre-cultures were diluted 1:100 in fresh THY medium and grown at 37° C., 5% CO 2 until OD 620 reached 0.3. Glycine was added to the medium to 10% final concentration and growth was maintained for an additional hour. Cells were spun down at 4° C. at 2500 ⁇ g and washed three times with electroporation buffer (5 mM KH 2 PO 4 , 0.4 M D-sorbitol, 10% glycerol, pH 4.5), finally suspended in the same buffer and equalized to the same OD 620 .
  • electroporation buffer 5 mM KH 2 PO 4 , 0.4 M D-sorbitol, 10% glycerol, pH 4.5
  • DNA manipulations including DNA preparation (QIAprep Spin MiniPrep Kit, Qiagen), PCR (Phusion® High-Fidelity DNA Polymerase, Finnzyme), DNA digestion (restriction enzymes, Fermentas). DNA ligation (T4 DNA ligase, Fermentas). DNA purification (QIAquick PCR Purification Kit, Qiagen) and agarose gel electrophoresis were performed according to standard techniques or manufacturers' protocols with some modifications (35). Site-directed mutagenesis was done using QuikChange II XL kit (Stratagene) or PCR-based mutagenesis (37).
  • Synthetic oligonucleotides (Sigma-Aldrich & Biomers) and plasmids used and generated in this study are listed in Supplementary Table S1. The integrity of all constructed plasmids was verified by enzymatic digestion and sequencing at LGC Genomics.
  • the backbone shuttle vector pEC85 was used for complementation study (38,39).
  • the RNase-III encoding genes (mc genes) of S. pyogenes, S. mutans, S. thermophilus, C. jejuni, N. meningitidis, P. multocida, F. novicida, E. coli and S. aureus and the genes encoding truncated and inactive RNase III variants (truncated and inactive (D51A) mc mutants) of S. pyogenes were cloned in pEC483 (pEC85 containing the native promoter of S.
  • pyogenes mc using NcoI and EcoRI restriction sites (Supplementary Table S1, Supplementary FIG. S6 ).
  • the ortholog and mutant cas9 genes were cloned in pEC342 (pEC85 containing a sequence encoding tracrRNA-171 nt (16) and the native promoter of the S. pyogenes cas operon) using SalI and SmaI restriction sites (Supplementary Table S1). Note that in a previous study, we observed low abundance of tracrRNA in the cas9 deletion mutant. For this reason, plasmids used in cas9 complementation studies were designed to encode tracrRNA in addition to cas9 (16).
  • the generated mc and cas9 recombinant plasmids were introduced in S. pyogenes ⁇ mc and ⁇ cas9 deletion strains, respectively (Supplementary Table S1). Plasmid integrity in all complemented strains was checked by plasmid DNA extraction and digestion.
  • Plasmid pEC85 was used as backbone vector for transformation studies.
  • a DNA fragment containing WT speM protospacer sequence was cloned in the PstI site of plasmids containing coding sequences of WT or mutated cas9 from S. pyogenes (Supplementary Table S1).
  • the overexpression vector pET16b (Novagen) was modified by inserting three additional restriction sites (SalI, SacI, NotI) into the NdeI restriction site, generating pEC621.
  • the genes coding for the orthologous Cas9 proteins were PCR amplified from genomic DNA of the corresponding strains using primers containing a SalI and a NotI restriction site (Supplementary Table S1).
  • the S. pyogenes cas9 mutant genes were PCR amplified from the complementation plasmids mentioned above. All orthologous and mutant cas9 genes were cloned into the SalI and NotI sites of pEC621.
  • Plasmid pEC287 that contains the speM protospacer sequence was used as a vector to construct all substrate plasmids.
  • the PAM sequence located in 3′ just next to the crRNA-targeted sequence of the speM protospacer (GGG on this plasmid) was modified by PCR-mediated site-directed mutagenesis (37) using one standard oligonucleotide (OLEC 3140 or OLEC3194) that either introduced or removed a XbaI restriction site for screening purposes, and a second mutagenic oligonucleotide to exchange the protospacer adjacent sequence (Supplementary Table S1).
  • RNA from S. pyogenes SF370 WT, deletion mutants and complemented strains was prepared from culture samples collected at the mid-logarithmic phase of growth using TRIzol (Sigma-Aldrich). The total RNA samples were treated with DNase I (Fermentas) according to the manufacturer's instructions. The concentration of RNA in each sample was measured using NanoDrop.
  • EDC 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
  • Oligonucleotide probes (40 pmol) were labeled with 32 P (20 ⁇ Ci) using the T4-polynucleotide kinase (10 U, Fermentas) and purified using G-25 columns (GE Healthcare) prior use. Visualization of the radioactive signal was done using a phosphorimager. 5S rRNA served as loading control.
  • E. coli Rosetta2(DE3) and E. coli NiCo21(DE3) were transformed with overexpression plasmids coding for S. pyogenes WT and mutant or orthologous Cas9, respectively.
  • Cells were grown at 37° C. to reach an OD 600 of 0.7-0.8, protein expression was induced by adding IPTG to a final concentration of 0.5 mM and cultures were further grown at 13° C. overnight. The cells were harvested by centrifugation and the pellet was resuspended in lysis-buffer (20 mM HEPES pH 7.5, 500 mM KCl [1 M for S.
  • thermophilus * Cas9] 0.1% Triton X-100, 25 mM imidazole
  • lysed by sonication The lysate was cleared by centrifugation (>20 000 ⁇ g) and incubated with Ni-NTA (Qiagen) for 1 h at 4° C.
  • the recombinant protein was eluted with elution-buffer (20 mM HEPES pH 7.5, 150 mM KCl, 0.1 mM DTT, 250 mM imidazole, 1 mM EDTA) and the fractions were analyzed by SDS-PAGE.
  • elution-buffer 20 mM HEPES pH 7.5, 150 mM KCl, 0.1 mM DTT, 250 mM imidazole, 1 mM EDTA
  • the protein containing eluates were pooled and further purified via HiTrap SP FF (GE Healthcare) cation-exchange chromatography.
  • the protein was loaded on the column equilibrated with buffer A (20 mM HEPES pH 7.5, 100 mM KCl) using an FPLC system (Akta, GE Healthcare).
  • Cas9 was eluted with a gradient of buffer B (20 mM HEPES pH 7.5, 1 M KCl) over 12 ml. 1 ml fractions were collected and analyzed by SDS-PAGE. The protein containing fractions were pooled and dialyzed overnight (20 mM HEPES pH 7.5, 150 mM KCl, 50% glycerol).
  • the eluates from Ni-NTA purification were checked for purity by SDS-PAGE.
  • RNA for in vitro DNA cleavage assays was generated by in vitro transcription using the AmpliScribeTM T7-FlashTM Transcription Kit (Epicentre) according to the manufacturer's instructions. PCR products or synthetic oligonucleotides used as templates are listed in Supplementary Table S1.
  • the synthesized tracrRNA and repeat region of crRNA from each bacterial species correspond to the mature forms of RNAs as determined by deep RNA sequencing (15) or bioinformatics predictions.
  • the spacer region of all crRNAs used in this study targets the speM protospacer (encoding superantigen; targeted by spacer 2 of S. pyogenes SF370 CRISPR array, Spyo1h_002 (16)).
  • RNAs were precipitated and further purified from 10% polyacrylamide 8 M urea denaturing gel. The RNA concentration was determined by measuring the OD 260 and the molarity was calculated. Equimolar amounts of crRNA and tracrRNA were mixed in 5 ⁇ RNA annealing buffer (1 M NaCl, 100 mM HEPES pH 7.5), heated up to 95° C. for 5 min and slowly cooled to room temperature before use.
  • cleavage assays using Cas9 mutant proteins 25 nM of Cas9 were incubated with equimolar amounts of prehybridized S. pyogenes dual-RNA in cleavage buffer (20 mM HEPES pH 7.5, 150 mM KCl, 10 mM MgCl 2 , 0.5 mM DTT, 0.1 mM EDTA) for 15 min at 37° C. Plasmid DNA (5 nM) containing speM (NGG PAM) was added and further incubated for 1 h at 37° C.
  • cleavage buffer 20 mM HEPES pH 7.5, 150 mM KCl, 10 mM MgCl 2 , 0.5 mM DTT, 0.1 mM EDTA
  • the reaction was stopped by addition of 5 ⁇ loading buffer (250 mM EDTA, 30% glycerol, 1.2% SDS, 0.1% (w/v) bromophenol blue) and analyzed by 1% agarose gel electrophoresis in 1 ⁇ TAE. Cleavage products were visualized by ethidium bromide staining. All other cleavage assays were carried out using the same conditions with the following modifications: KGB (43) (100 mM potassium glutamate, 25 mM Tris/acetate pH 7.5, 10 mM Mg-acetate, 0.5 mM 2-mercaptoethanol, 10 ⁇ g/ml BSA) was used as cleavage buffer and different concentrations of dual-RNA:Cas9 complex were analyzed. The concentration of plasmid DNA was kept constant in all experiments, i.e. 5 nM.
  • 5 ⁇ loading buffer 250 mM EDTA, 30% glycerol, 1.2% SDS, 0.1% (w/v) bromophenol blue
  • Spacer sequences of the selected bacterial species were extracted from the CRISPRdatabase (http://crispr.u-psud.fr/crispr/) and used to find cognate protospacer candidates using megaBLAST (http://blast.ncbi.nih.gov/Blast).
  • Protospacer candidates were defined as containing a sequence with ⁇ 90% similarity to the crRNA spacer sequence and originating from phage, plasmid or genomic DNA related to the bacterial species of the targeting CRISPR-Cas.
  • the orientation of transcription was determined previously by RNA sequencing or Northern blot analysis (15,16).
  • the PAM sequence is located in 3′ of the protospacer, juxtaposed to the sequence targeted by cognate crRNA on the non-target strand (14,18,23,44).
  • 10 nt sequences on the non-target strand directly downstream of each protospacer sequence were aligned.
  • a logo plot http://weblogo.berkeley.edu/ showing the most abundant nucleotides was created and PAM sequences were predicted.
  • CRISPR-Cas loci for which no suitable protospacer sequences could be identified S. mutans UA159, C. jejuni NCTC 11168, P. multocida Pm70, F.
  • Position-Specific Iterated (PSI)-BLAST program 45 was used to retrieve orthologs of the Cas9 family in the NCBI nr database. Sequences shorter than 800 amino acids were discarded.
  • the BLASTClust program (46) set up with a length coverage cutoff of 0.8 and a score coverage threshold (bit score divided by alignment length) of 0.8 was used to cluster the remaining sequences (Supplementary Table S2). This procedure produced 82 clusters.
  • sequences reported in this study one or several representatives from each cluster were selected and aligned using the MUSCLE program (47) with default parameters, followed by a manual correction on the basis of local alignments obtained using PSI-BLAST (45) and HHpred programs (48).
  • the confidently aligned blocks (Supplementary FIG. S2 ) with 285 informative positions were used for maximum likelihood tree reconstruction using the FastTree program (49) with the default parameters: JTT evolutionary model, discrete gamma model with 20 rate categories.
  • the same program was used to calculate the bootstrap values.
  • Cas1 sequences were selected from the corresponding cas operons (Supplementary Table S2). A few incomplete sequences were substituted by other Cas1 sequences from the same Cas9 cluster (Supplementary Table S2).
  • Several Cas1 proteins from subtypes I-A, B, C and E were included as an outgroup.
  • Cas1 sequences were aligned using the same approach described above and 252 informative positions (Supplementary FIG. S3 ) were used for maximum likelihood tree reconstruction using the FastTree program.
  • RNase III multiple sequence alignment was prepared using the MUSCLE program.
  • RNA duplex secondary structures were predicted using RNAcofold of the Vienna RNA package (50,51) and RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/rnahvbrid/). The structure predictions were then visualized using VARNA (52).
  • pyogenes SF370 tracrRNA OLEC101 GGACTAGCCTTATTTTAACTTG R NB (3′ probe) 4 crRNA (CRISPR01 (type II-A) expression in S.
  • pyogenes SF370 crRNA OLEC104 GGACCATTCAAAACAGCATAGCTCTAAAAC R NB (repeat) 9 Loading controls for Northern blots 5S rRNA OLEC288 CTAAGCGACTACCTTATCTCA R NB His-tagged cas9 constructs (pEC85-based) pEC710 OLEC215 GCAG GAATTC ATCAGTGATGGTGATGGTGATGCCCGGGTT F Cloning 1 TGTCGACCT CCTAAAATAAAAAGTTTAAATTAAATC OLEC206 GGTGGT CTGCAG GTTTGCAGTCAGAGTAGAATAGAAG R 6 pEC714 OLEC209 ATGCAG GTCGAC ATGGATAAGAAATACTCAATAGGC F Expression 6 cs
  • Cas9 orthologs were identified in 653 bacterial strains representing 347 species (Supplementary Table S2). After removing incomplete or highly similar sequences, we selected 83 diverse, representative Cas9 orthologs for multiple sequence alignment and phylogenetic tree reconstruction ( FIG. 1A , Supplementary Table S2, Supplementary FIGS. S2 and S 4 , see Materials and Methods). The Cas9 tree topology largely agrees with the phylogeny of the corresponding Cas1 proteins (Supplementary Table S2, Supplementary FIGS.
  • Type II CRISPR-Cas into three subtypes, II-A (specified by csn2), II-B (characterized by long and most diverged cas9 variants (formerly csx12) and cas4), and II-C(three-cas gene operon) (15).
  • Cas9 length Cluster a Strain b (aa) Cas9 GI Cas1GI c Subtype d 1 Dolosigranulum pigrum ATCC 51524 1332 375088882 Type II-A Enterococcus faecalis ATCC 29200 1337 229548613 Enterococcus faecalis ATCC 4200 1337 256617555 Enterococcus faecalis D6 1337 257086028 Enterococcus faecalis E1Sol 1337 257080914 Enterococcus faecalis OG1RF 1337 384512368 Enterococcus faecalis TX0470 1337 312900261 Enterococcus faecalis TX4244 1337 422695652 Enterococcus faecium 1,141,733 1339 257888853 Enterococcus fae
  • constellatus 1345 418965022 SK53 Streptococcus dysgalactiae subsp. equisimilis 1371 410494913 AC-2713 Streptococcus dysgalactiae subsp. equisimilis 1371 386317166 ATCC 12394 Streptococcus dysgalactiae subsp. equisimilis 1371 251782637 GGS_124 Streptococcus dysgalactiae subsp. equisimilis 1371 408401787 RE378 Streptococcus equi subsp.
  • zooepidemicus 1348 195978435 MGCS10565 Streptococcus equinus ATCC 9812 1377 320547102 Streptococcus gallolyticus subsp. gallolyticus 1370 325978669 ATCC BAA-2069 Streptococcus gallolyticus subsp. gallolyticus 1370 306831733 TX20005 Streptococcus gallolyticus UCN34 1371 288905639 Streptococcus infantarius subsp.
  • jejuni 2008-2025 987 419650041 Campylobacter jejuni subsp. jejuni 2008-872 984 419654778 Campylobacter jejuni subsp. jejuni 2008-979 987 419660762 Campylobacter jejuni subsp. jejuni 2008-988 965 419656328 Campylobacter jejuni subsp. jejuni 2008-988 984 419655317 Campylobacter jejuni subsp. jejuni 260.94 961 86152042 Campylobacter jejuni subsp. jejuni 414 985 283953849 Campylobacter jejuni subsp.
  • jejuni ATCC 33560 984 419643715 Campylobacter jejuni subsp. jejuni CF93-6 987 86149266 Campylobacter jejuni subsp. jejuni CG8486 984 148925683 Campylobacter jejuni subsp. jejuni HB93-13 984 86152450 Campylobacter jejuni subsp. jejuni LMG 23210 987 419696801 Campylobacter jejuni subsp. jejuni LMG 23211 984 419697443 Campylobacter jejuni subsp. jejuni LMG 23263 984 419628620 Campylobacter jejuni subsp.
  • F0487 1450 393778597 Chryseobacterium sp. CF314 1419 399023756 Fibrobacter succinogenes subsp. succinogenes 1512 261414553 S85 Flavobacteriaceae bacterium S85 1516 372210605 Flavobacterium columnare ATCC 49512 1459 365960762 Fluviicola taffensis DSM 16823 1458 327405121 Mucilaginibacter paludis DSM 18603 1473 373954054 Myroides odoratus DSM 2801 1466 374597806 Omithobacterium rhinotracheale DSM 15997 1535 392391493 Prevotella bivia JCVIHMP010 1485 282858617 Prevotella buccae ATCC 33574 1457 315607525 Prevotella nigrescens ATCC 33563 1506 340351024 Prevotella sp.
  • AK2 1345 410030899 Myroides injenensis M09-0166 1401 399927444 Niabella soli DSM 19437 1426 374372722 Parabacteroides johnsonii DSM 18315 1443 218258638 Parabacteroides sp. D13 1424 256840409 Prevotella histicola F0411 1375 357042839 Prevotella intermedia 17 1380 387132277 Prevotella nigrescens F0103 1380 445119230 Prevotella oralis ATCC 33269 1391 323344874 Prevotella sp. oral taxon 306 str.
  • JGS1495 1065 169343975 Clostridium perfringens D str. JGS1721 1065 182624245 Sporolactobacillus vineae DSM 21990 SL153 1084 404330915 10 Gemella haemolysans ATCC 10379 1392 241889924 Type II-A Gemella morbillorum M424 1385 317495358 Megasphaera sp. UPII 135-E 1352 342218215 Veillonella atypica ACS-134-V-Col7a 1398 303229466 303229394 Veillonella parvula ATCC 17745 1398 282849530 Veillonella sp.
  • holarctica LVS 158 89256630 Francisella tularensis subsp.
  • holarctica LVS 393 89256631 Francisella tularensis subsp.
  • holarctica URTF1 53 290953529 Francisella tularensis subsp.
  • holarctica URTF1 285 290953528 Francisella tularensis subsp.
  • oral taxon 317 str. F0108 593 288929745 Prevotella sp. oral taxon 317 str.
  • Cas9 sequences are grouped according to the BLASTclust clustering program. Truncated sequences were not selected for the analysis and are listed at bottom of the table without any cluster number (see Materials and Methods).
  • b Bacterial strains harboring cas9 gene orthologue are listed; GI, GenInfo Identifier. Bold, cluster representatives chosen for the alignment and tree reconstruction. Grey, discarded, incomplete Cas9 sequences (see Materials and Methods). Note, that the incomplete sequences were all confirmed to be truncated Cas9 orthologues due to the presence of conserved motifs and similarity to the other Cas9 orthologues.
  • c Cas1 GenInfo Identifier of the representative sequences chosen for the alignment and tree reconstruction are given. Grey, discarded, incomplete sequences. When possible, alternative Cas1 sequence from the same cluster as the discarded Cas1 sequence was selected (clusters 8, 9 and 21, in bold). d Type II CRISPR subtype of the CRISPR loci of the Cas9 cluster as inferred from the representative Cas1 and Cas9 trees topology.
  • Cas9 enzymes representative of the major type II groups.
  • Cas9 orthologs of S. pyogenes, S. thermophilus * (CRISPR3) and S. mutans were chosen for type II-A systems associated with shorter, ⁇ 220 amino acid Csn2 variants (Csn2a).
  • thermophilus ** represents a distinct group of type II-A sequences associated with longer, ⁇ 350 amino acid version of Csn2 orthologs (Csn2b).
  • Cas9 of F. novicida was selected for type II-B.
  • the closely related Cas9 orthologs of P. multocida and N. meningitidis and the distinct, short Cas9 of C. jejuni were chosen for type II-C ( FIG. 1B ).
  • Expression of associated tracrRNAs and crRNAs in S. pyogenes, S. mutans, F. novicida, N. meningitidis and C. jejuni was already validated by deep RNA sequencing (15,16). The RNAs in S.
  • thermophilus and P. multocida were predicted bioinformatically based on the sequences from related species within the same type II group.
  • FIG. 1B shows the organization of the eight selected type II CRISPR-Cas loci and highlights our previous findings demonstrating that the type II loci architectures are highly variable among subtypes, yet conserved within each group (15). These variations are in good agreement with the clustering derived from the Cas9 and Cas1 phylogenetic trees ( FIG. 1A , Supplementary FIG. S4 ).
  • Strains were also isolated from invertebrates and environmental samples, including fresh and sea water, plant material, soil and food, the latter comprising species used in fermentation processes. Cas9 is also present in species from extreme environments such as deep sea sediments, hot springs and Antarctic ice, further demonstrating the wide spread of type II CRISPR-Cas systems in bacteria. A comparison of the taxonomy and habitats of representative strains with the phylogenetic clustering of Cas9 sequences shows little correlation (Supplementary FIG. S11 ). In particular, clusters of Cas9 genes were identified from taxonomically distant bacteria that were isolated from similar habitats.
  • Examples include diverse Firmicutes, Molicutes, Spirochaete and Fusobacteria, that were all isolated from gastrointestinal tracts of mammals, and members of different Proteobacteria, Firmicutes and Fusobacteria families mostly found in environmental samples (Supplementary FIG. S11 , clusters 1 and 3). A few exceptions involve grouping of Cas9 genes from closely related species isolated from diverse habitats such as Actinobacteria isolated from human and dog specimens but also from hot springs (Supplementary FIG. S11 , clusters 2, 4 and 5). This complex distribution of Cas9 across bacterial genomes indicates that evolution of dual-RNA:Cas9 systems in bacteria occurs both vertically and horizontally (55).
  • RNase III plays an essential role in the biogenesis of dual-RNA:Cas9 systems by co-processing tracrRNA and pre-crRNA at the level of antirepeat:repeat duplexes (16,17).
  • the interchangeability of S. pyogenes RNase III with RNases III from selected bacterial species was analyzed in the co-processing of S. pyogenes tracrRNA:pre-crRNA, including strains that lack type II CRISPR-Cas ( S. aureus COL, E. coli TOP10).
  • Northern blot analysis showed that all RNases III studied can co-process the RNA duplex ( FIG. 2 , Supplementary FIG.
  • Cas9 HNH and Split RuvC Domains are the Catalytic Moieties for DNA Interference
  • FIG. 3A To investigate the relevance of all catalytic motifs for tracrRNA:pre-crRNA processing and/or DNA interference, alanine substitutions of selected residues were created ( FIG. 3A ).
  • Cas9 point mutants of conserved amino acid residues in the central RuvC motifs (14) FIG. 3A , Supplementary FIG. S2 .
  • Northern blot analysis of S. pyogenes cas9 deletion mutant complemented with each of the cas9 point mutants revealed the presence of mature tracrRNA and crRNA forms, demonstrating that none of the catalytic motifs is involved in dual-RNA maturation by RNase III.
  • Dual-RNA and Cas9 sequences have widely evolved in bacteria (15). However, despite the high sequence variability among Cas9 sequences, certain motifs are conserved. In addition to the previously identified central HNH and N-terminal RuvC catalytic motifs (20,21,44,56), we show that the two middle RuvC motifs are required for interference activity in vivo and in vitro. In agreement with previous findings, deactivation of either one of the catalytic motifs (RuvC or HNH) results in nicking activity of Cas9 originating from the other motif (2,8,24,25). None of the mutations introduced in these conserved motifs affected the role of Cas9 in tracrRNA:pre-crRNA maturation by RNase III in vivo.
  • thermophilus * can substitute for the stabilizing role of S. pyogenes Cas9 in RNA processing by RNase III ( FIG. 4B , Supplementary FIG. S8 ).
  • Cas9 from S. thermophilus**, C. jejuni, N. meningitidis, P. multocida and F. novicida could not complement the lack of RNA processing in the cas9 mutant of S. pyogenes .
  • the 75-nt processed form of tracrRNA is observed as a very weak signal of background level of dual-RNA processed by RNase III in the absence of Cas9.
  • Cas9 from closely related systems of S. pyogenes in the type II-A cluster can substitute endogenous Cas9 role in dual-RNA stabilization and subsequent maturation by RNase III.
  • substitution of orthologs from the selected species for the endogenous S. pyogenes Cas9 shows that only Cas9 proteins from the S. pyogenes subcluster are capable of assisting tracrRNA:pre-crRNA processing by RNase III. This result indicates that the less-conserved inter-motif regions, which are the basis for the Cas9 subgrouping, could be responsible for Cas9 specificity for certain dual-RNAs.
  • strains that harbor a closely related variant of Cas9 (Supplementary Table S2) were searched and their spacer sequences analyzed following the same approach (Supplementary Table S3).
  • the identified 10 nt sequences located directly downstream of the protospacer sequence were aligned and the most common nucleotides that could represent PAM sequences were delineated.
  • plasmid DNA substrates were designed containing the speM protospacer followed by different adjacent sequences either comprising the predicted PAM or not ( FIG. 5B ).
  • the Cas9 orthologous proteins were purified (Supplementary FIG.
  • NNNNGATT meningitidis
  • S. thermophilus * dual-RNA guided Cas9 from S. thermophilus * could efficiently cleave target DNA in the presence of only NGG instead of NGGNG (Supplementary FIG. S9 ). This is in contrast to data obtained in vivo, where mutation of the third G abrogates interference by Cas9 of S. thermophilus * (23).
  • S. thermophilus ** the PAM was published as NNAGAAW (27), which differs by one base from the sequence that we derived (NNAAAAW).
  • CRISPR-Cas spacer sequences from selected bacterial strains with BLAST candidate protospacers and their downstream sequence.
  • Number of CRISPR Strain a spacers Spacer b Spacer sequence Sreptococcus pyogenes 6 1 TGCGCTGGTTGATTTCTTCTTGC SF370 GCTTTTT (Accession: NC_002737) 2 TTATATGAACATAACTCAATTTG TAAAAAA 3 AGGAATATCCGCAATAATTAATT GCGCTCT 4 AGTGCCGAGGAAAAATTAGGTGC GCTTGGC 5 TAAATTTGTTTAGCAGGTAAACC GTGCTTT Streptococcus mutans 5 3 CTAACTATGATGACACAACAGCT UA159 (Accession: NC_004350) TTTAGCG Streptococcus mutans LJ23 8 2 TGAAGTGCAAGCTTACGTGACTG (Accession: NC_017768) ACTCGCG Streptococcus mutans
  • jejuni 5 1-5 NCTC 11168 (Accession: NC_002163) Campylobacter jejuni 5 3 TCATCATCACTTAAAACCTTAAA subsp. jejuni CF93-6 TTTACC (Accession: AANJ00000000) Campylobacter jejuni subsp. jejuni 9 1 GCATTGCTTTACTACATAGCCAG HB93-13c_jejuni_subsp_jejunihb_13_42 TCGTGTA (Accession: AANQ00000000) Campylobacter jejuni subsp.
  • jejuni 1336 1 TTACTGATATTAAAATTAACTCC (Accession: NZ_CM000854 ATAATTT NZ_ADGL01000000) 2 ATAAAGCTAATGCAAAAGTTGAA AACAAA Campylobacter jejuni subsp.
  • TGAGTGA (Accession: NZ_CM000855 NZ_ADGM01000000) Neisseria meningitidis 16 2 CTTCTGCCTTTTTACAAGCTCGC serogroup A TTTCTTT strain Z2491 3 TTTGGTAAAGGTTTCTGTTGCGA (Accession: NC_003116) CCCGAAT 7 AAATTCGTTTCAGATAGCAAACG CAGTAGT 12 GGGTAGCCAGTGCTAAAACCGCA CCCGCTT 13 CCAAATAGAAATACATACGCCGA GTAATTA 14 TTTCTTTGTAATTGTTCTGCC TTTTA 15 TACCCACGGCGGAAACCATTGCC ACAAAAC Pasteurella multocida 5 1-5 str.
  • pyogenes MGAS1882 (MGAS1882_1116), MGAS8232 (spyM18_0769), MGAS10394 (M6_Spy0995, 100 (Accession: NC_002737) M6_Spy1349), SSI-1 (SPs0926), ⁇ P9 endopeptidase gene S.
  • pyogenes MGAS2096 (MGAS2096_Spy1450), A20 (A20_1472c), M1 476 (M1GAS476_1503), MGAS9429 97 (MGAS9429_Spy1426), MGAS5005 (M5005_Spy1424) endopeptidase gene S.
  • pyogenes M1 GAS (SPy_0700), MGAS2096 (MGAS2096_Spy0592) 97 endopeptidase gene S.
  • pyogenes MGAS6180 (M28_Spy1234); NIH1 (NIH1.1_43), SSI-1 (SPs0647), MGAS315 (SpyM3_0930, 100 SpyM3_1215) phage related gene gene for pyrogenic exotoxin M (speM) of several Streptococci strains 100 S.
  • pyogenes MGAS8232 (spyM18_0742), MGAS10750 (MGAS10750_Spy0588), MGAS10270 100 (MGAS10270_Spy0563) adenine specific methylase gene S.
  • pyogenes Manfredo (SpyM50653) adenine specific methylase gene 97 S.
  • MGAS10750 (MGAS10750_Spy0839), MGAS10270 (MGAS10270_Spy0546, MGAS10270_Spy0804), SSI-1 (SPs0517, SPs0888), 100 MGAS1882 (MGAS1882_1156), MGAS8232, NZ131(Spy49_1511c), MGAS315 (SpyM3_0965, SpyM3_1347) phage protein gene or intergenic region Streptococcus mutans UA159 (Accession: NC_004350) ⁇ M102 (orf13) putative tail protein gene 100 Streptococcus mutans LJ23 (Accession: NC_017768) ⁇ M102 (orf15) putative minor structural protein 90 Streptococcus mutans GS-5 (Accession: NC_018089) ⁇ M102 (orf15) putative minor structural protein 97 ⁇ M102 100 ⁇ M
  • jejuni NCTC 11168 (Accession: NC_002163) Campylobacter jejuni subsp. jejuni CF93-6 (Accession: AANJ00000000)
  • C. jejuni RM1221 CJE1445) hypothetical protein gene 93 Campylobacter jejuni subsp. jejuni HB93-13c_ jejuni _subsp_jejunihb_13_42 (Accession: AANQ00000000) C. jejuni subsp. doylei 269.97 (JJD26997_1148) conserved hypothetical protein gene 100 Campylobacter jejuni subsp. jejuni NW C. jejuni subsp.
  • doylei 269.97 JJD26997_0867 putative primase gene 97 genomic scaffold Mich_State_Univ:Contig3 (Accession: JH376989 C. jejuni subsp. jejuni PT14 (A911_r08426, A911_r08428, A911_r08430), NCTC 11168-BN148 (BN148_r02, BN148_r05, BN148_r08), S3 (CJS3_1811, CJS3_1817, 100 REGION: 13521 . . .
  • CJS3_1830 ICDCCJ07001 (ICDCCJ07001_29, ICDCCJ07001_396, ICDCCJ07001_718), M1 (CJM1_0031, CJM1_0413, CJM1_0727), IA3902 (CJSA_Cj23SA, CJSA_Cj23SB, CJSA_Cj23SAC), BABS091400, 81116 (C8J_Cj23SA, C8J_Cj23SB, C8J_Cj23SC), 81-176 (CJJ81176_1714, CJJ81176_1727, CJJ81176_1707), NCTC 11168; C.
  • jejuni S3 CJS3_0723
  • ⁇ CGC-2007 prophage related genes 100 NZ_CM000854 NZ_ADGL01000000
  • C. jejuni NCTC 13255 (putative CJIE1-2-like prohage), 99-7046 (putative CJIE1-3-like prophage), 00-2425 (putative CJIE1 prophage), RM1221 (CJE0227)
  • jejuni NCTC 13255 (putative CJIE1-2-like prophage), 99-7046 (patative CJIE1-3-like prophage), 00-3477 (putative CJIE1-4 Mu-like prophage), 00-2425 (putative 100 CJIE1 prophage), RM1221 (CJE0238), C. jejuni subsp. jejuni S3 (CJS3_0704), ICDCCJ07001, C. hyoilei hypothetical protein gene Campylobacter jejuni subsp. jejuni 414 (Accession: NZ_CM000855 C. jejuni subsp.
  • jejuni RM1221 CJE0778
  • C. jejuni subsp. doylei 269.97 JJD26997_1327) excinuclease ABC subunit B gene Neisseria meningitidis serogroup A strain Z2491 N. gonorrhoeae (NGU65994, PivNG), FA 1090 (NGO1137, NGO1164, NGO1262) invertase related genes, phage associated protein genes 97 (Accession: NC_003116) N.
  • meningitidis NZ-05/33 (NMBNZ0533_1722), M04- 240196 (NMBNZ0533_1722), M01-240149 (NMBH4476_1701), H44/76 (NMBH4476_1701) 100 hypothetical proteins upstream of transposase gene
  • N. lactamica isolate 3207487 (plasmid pNL3.2), N. lactamica (plasmid pNL9)
  • plasmid pNL9 97 N. gonorrhoeae TCDC-NG08107, NCCP11945 intergenic region (putative phage proteins)
  • 93 N. gonorrhoeae NCCP11945 (NGK_1948, NGK_1990, NGK_2023) hypothetical protein genes 93 N.
  • gonorrhoeae intergenic region PivNG 93 N. gonorrhoeae FA 1090 numerous intergenic regions in prophages 93 N. gonorrhoeae TCDC-NG08107, N. gonorrhoeae NCCP11945 intergenic region (putative phage proteins) 97 N. lactamica plasmid pNL9 93 N. meningitidis plasmid pJS-B 100 N. lactamica plasmid pNL9 93 N. meningitidis plasmid pJS-B 97 N. lactamica plasmid pNL9 100 N. meningitidis plasmid pJS-B 100 N.
  • N. meningitidis serogroup C 100 FAM18 (NMC1864) hypothetical protein gene
  • N. meningitidis M04-240196 NMBM04240196_0048, NMBM04240196_0749
  • putative membrane protein gene 100 Pasteurella multocida no significant BLAST hits str.
  • Pm70 (Accession: NC_002663) Pasteurella multocida subsp. gallicida X73
  • P. multocida 1.8 kb plasmid 100 (Accession: CM001580 AMBP01000000) P.
  • novicida 3523 (FN3523_1009) phage-related baseplate assembly protein gene 89 GA99-3548 supercont1.3
  • F. cf. novicida 3523 (FN3523_1006) hypothetical protein gene 94 (Accession: DS264589 ABAH01000000)
  • F. cf. novicida 3523 (FN3523_0999) hypothetical protein gene 91 a Selected strains used in this study. No potential protospacers were found for Streptococcus mutans UA159, Campylobacter jejuni subsp. jejuni NCTC 11168, Pasteurella multocida str. Pm70 and Francisella tularensis subsp. novicida U112.
  • SUPPLEMENTARY TABLE S4 Cas9 is present in bacteria from 12 different phyla and diverse habitats Strain a Class Isolation/habitat b Actinobacteria Actinobacteridae Acidothermus cellulolyticus 11B Acidothermaceae extremophile (hot water spring) Actinomyces coleocanis Actinomycetaceae dog genital tract Actinomyces georgiae F0490 Actinomycetaceae oral cavity Actinomyces naeslundii str. Howell 279 Actinomycetaceae oral cavity Actinomyces sp. ICM47 Actinomycetaceae ND Actinomyces sp. oral taxon 175 str.
  • F0384 Actinomycetaceae oral cavity Actinomyces sp. oral taxon 180 str.
  • F0310 Actinomycetaceae oral cavity Actinomyces sp. oral taxon 181 str.
  • F0379 Actinomycetaceae oral cavity Actinomyces sp. oral taxon 848 str.
  • F0450 Porphyromonadaceae oral cavity Prevotella bivia JCVIHMP010 Prevotellaceae genital tract Prevotella buccae ATCC 33574 Prevotellaceae oral cavity Prevotella buccalis ATCC 35310 Prevotellaceae oral cavity Prevotella denticola F0289 Prevotellaceae oral cavity Prevotella disiens FB035-09AN Prevotellaceae oral cavity Prevotella histicola F0411 Prevotellaceae oral cavity Prevotella intermedia 17 Prevotellaceae oral cavity Prevotella melaninogenica D18 Prevotellaceae oral cavity/rumen Prevotella micans F0438 Prevotellaceae oral cavity Prevotella multiformis DSM 16608 Prevotellaceae oral cavity Prevotella nigrescens ATCC 33563 Prevotellaceae oral cavity Prevotella oralis ATCC 33269 Prevotellaceae oral cavity Prevotella oulor
  • C561 Prevotellaceae oral cavity Prevotella sp. MSX73 Prevotellaceae oral cavity Prevotella sp. oral taxon 306 str.
  • F0472 Prevotellaceae oral cavity Prevotella sp. oral taxon 317 str.
  • F0108 Prevotellaceae oral cavity Prevotella sp. oral taxon 472 str.
  • Prevotellaceae oral cavity Prevotella stercorea DSM 18206 Prevotellaceae gastrointestinal tract/feces Prevotella tannerae ATCC 51259 Prevotellaceae oral cavity Prevotella timonensis CRIS 5C-B1 Prevotellaceae wound (breast abscess) Prevotella veroralis F0319 Prevotellaceae oral cavity Tannerella sp.
  • Flavobacteriaceae extremophile deep sea sediment
  • Flavobacteriaceae environmental sample Kordia algicida OT-1
  • Flavobacteriaceae environmental sample Myroides injenensis M09-0166
  • Flavobacteriaceae human clinical specimens Myroides odoratus DSM 2801
  • Flavobacteriaceae fish Flavobacteria (continued) Omithobacterium rhinotracheale DSM 15997
  • Flavobacteriaceae extremophile (antarctic ice)
  • Riemerella anatipesfifer ATCC 11845 DSM 15868
  • Lactobacillaceae food (fermented) Lactobacillus farciminis KCTC 3681 Lactobacillaceae food (fermented) Lactobacillus fermentum ATCC 14931 Lactobacillaceae food (fermented) Lactobacillus florum 2F Lactobacillaceae vegetation Lactobacillus gasseri JV-V03 Lactobacillaceae oral cavity Lactobacillus hominis CRBIP 24.179 Lactobacillaceae gastrointestinal tract/feces Lactobacillus iners LactinV 11V1-d Lactobacillaceae genital tract/urine Lactobacillus jensenii 269-3 Lactobacillaceae genital tract/blood Lactobacillus johnsonii DPC 6026 Lactobacillaceae pig gastrointestinal tract Lactobacillus mucosae LM1 Lactobacillaceae wild pig gastrointestinal tract Lactobacillus paracasei subsp
  • Lactobacillaceae food (fermented) Lactobacillus pentosus IG1 Lactobacillaceae food (fermented) Lactobacillus plantarum ZJ316 Lactobacillaceae gastrointestinal tract/feces Lactobacillus rhamnosus GG Lactobacillaceae gastrointestinal tract/feces Lactobacillus ruminis ATCC 25644 Lactobacillaceae rumen Lactobacillus salivarius UCC118 Lactobacillaceae oral cavity Lactobacillus sanfranciscensis TMW 1-1304 Lactobacillaceae food (fermented) Lactobacillus sp.
  • D21 Acidaminococcaceae gastrointestinal tract/feces Aminomonas paucivorans DSM 12260 Syntrophoomonadaceae environmental sample (sewage) Anaerococcus tetradius ATCC 35098 Peptostreptococcaceae human clinical specimens Butyrivibrio fibrisolvens 16/4 Lachnospiraceae rumen Catenibacterium mitsuokai DSM 15897 Lachnospiraceae gastrointestinal tract/feces Clostridium cellulolyticum H10 Clostridiaceae vegetation (composted) Clostridia (continued) Clostridium perfringens D str.
  • BTAi1 Bradyrhizobiaceae vegetation Caenispirillum salinarum AK4 Rhodospirillaceae extremophile (solar saltern) Dinoroseobacter shibae DFL 12 Rhodobacteraceae environmental sample (seawater) Gluconacetobacter diazotrophicus PAI5 Acetobacteriaceae vegetation Maritimibacter alkaliphilus ATCC2654 Rhodobacteraceae environmental sample (seawater) Methylocystis sp.
  • Methylocystaceae environmental sample (sewage, fresh water) Methylosinus trichosporium OB3b Methylocystaceae environmental sample (soil, fresh water) Nitrobacter hamburgensis X14 Bradyrhizobiaceae environmental sample (soil) Parvibaculum lavamentivorans DS-1 Phyllobacteriaceae environmental sample (sewage) Puniceispirillum marinum IMCC1322 SAR16 Glade environmental sample (seawater) Rhodopseudomonas palustris BisB18 Bradyrhizobiaceae environmental sample (soil) Rhodospirillum rubrum ATCC 11170 Rhodospirillaceae environmental sample (sea mud) Rhodovulum sp.
  • PH10 Rhodobacteraceae environmental sample (soil) Sphingobium sp.
  • S17 Sphingomonadaceae environmental sample (stromatolite) Tistrella mobilis KA081020-065 Rhodospirillaceae environmental sample (seawater) Betaproteobacteria Acidovorax avenae subsp.
  • Torquens KCTC 3535 Alicyclobacillus 53 GCGAGGGAUAUCAUACC GUCAUAGUUCCCUCACA 54 105 36 hesperidum ACAUCAAGGCUUGCGAG AGCCUCGAUGUGGUAUG URH17-3-68 GUUGCUAUGAUAAGGCA AU ACAGGCCGCAAAGCACU (SEQ ID NO: 2818) GACCCGCAUUCCAAUGA AUGCGGGUCAUCUACUU UUU (SEQ ID NO: 2747) Roseburia 52 None — — — inulinivorans DSM 16841 Uncult.delta 54 none GUCCUAGUUUCCCUUCC 8 — 36 proteobact.

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